Protein proximity assay in formalin fixed paraffin embedded tissue using caged haptens

ABSTRACT

Disclosed herein are caged haptens and caged hapten-antibody conjugates useful for enabling the detection of targets located proximally to each other in a sample.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 16/284,098 filed on Feb. 25, 2021, which is a continuation ofInternational Application No. PCT/US2017/48687 filed on Aug. 25, 2017,which claims the benefit of the filing date of International ApplicationNo. PCT/US2016/049153 filed on Aug. 26, 2016 and which applicationclaims the benefit of U.S. Provisional Patent Application No. 62/301,489filed Feb. 29, 2016, and the benefit of the filing date of U.S.Provisional Patent Application No. 62/211,590 filed Aug. 28, 2015; thepresent application is also is a continuation of U.S. patent applicationSer. No. 16/284,098 filed on Feb. 25, 2021 which is acontinuation-in-part of U.S. patent application Ser. No. 15/907,479,filed on Feb. 28, 2018, which is a continuation of InternationalApplication No. PCT/US2016/049153 filed on Aug. 26, 2016, and whichapplication claims the benefit of U.S. Provisional Patent ApplicationNo. 62/301,489 filed Feb. 29, 2016, and the benefit of the filing dateof U.S. Provisional Patent Application No. 62/211,590 filed Aug. 28,2015; the disclosures of each of the above-identified patentapplications are hereby incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

Disclosed embodiments concern detecting targets in a sample, includingtargets located proximally in a sample. Disclosed embodiments alsoprovide for a proximity assay for detecting protein dimers informalin-fixed, paraffin embedded tissue using caged haptens or cagedhapten conjugates.

STATEMENT OF INDUSTRIAL APPLICABILITY

The present disclosure has industrial applicability in the fields ofchemistry and diagnostics.

BACKGROUND OF THE INVENTION

Immunohistochemistry (IHC) refers to the processes of detecting,localizing, and/or quantifying antigens, such as a protein, in abiological sample using antibodies specific to the particular antigens.IHC provides the substantial advantage of identifying exactly where aparticular protein is located within the tissue sample. It is also aneffective way to examine the tissues themselves. In situ hybridization(ISH) refers to the process of detecting, localizing, and quantifyingnucleic acids. Both IHC and ISH can be performed on various biologicalsamples, such as tissue (e.g. fresh frozen, formalin fixed, paraffinembedded) and cytological samples. Recognition of the targets can bedetected using various labels (e.g., chromogenic, fluorescent,luminescent, radiometric), irrespective of whether the target is anucleic acid or an antigen. To robustly detect, locate, and quantifytargets in a clinical setting, amplification of the recognition event isdesirable as the ability to confidently detect cellular markers of lowabundance becomes increasingly important for diagnostic purposes. Forexample, depositing at the marker's site hundreds or thousands of labelmolecules in response to a single antigen detection event enhances,through amplification, the ability to detect that recognition event.

Networks of protein-protein interactions are the hallmarks of biologicalsystems. Protein-protein interactions form signal pathways that regulateall aspects of cellular functions in normal and cancerous cells. Methodshave been developed for detecting protein-protein interactions, such astransient receptor tyrosine kinase dimerization and complex formationafter extracellular growth factor activation; however, these methods arenot particularly designed to be used on formalin fixed paraffin embedded(FFPE) tissues.

The ability to interrogate for presence and distribution of specificintermolecular interactions for biomarkers known to be importantdeterminants in cancer biology is of high interest in the context of newdiagnostic capabilities and for determining therapeutic effect in thecontext of pharmaceutical development. The ability to probe and documentdistributions of molecular interactions on frozen and paraffin embeddedtissue has remained inaccessible; alternative technologies to approachthis question have been proposed, although the solutions have not provento be effective and reliable under practical use.

A proximity ligation assay has recently been developed by OLink AB. Thisis the only known commercial product for in situ detection ofprotein-protein interactions on formalin fixed paraffin embedded tissue.Proximity ligation assay technology uses DNA ligases to generate apadlock circular DNA template, as well as Phi29 DNA polymerase forrolling circle amplification. These enzymes are expensive. Moreover,these enzymes are not amenable for use with automated systems andmethods. For these reasons, proximity ligation assays are not consideredgenerally useful for commercial applications.

BRIEF SUMMARY OF THE INVENTION

Applicants have developed a superior method of detecting targets locatedproximally in a sample. In one aspect of the present disclosure is amethod for analyzing a sample to determine whether a first target isproximal to a second target, the method comprising: (a) forming a secondtarget-unmasking enzyme-antibody conjugate complex; (b) forming a firsttarget-caged hapten-antibody conjugate complex; (c) unmasking the cagedhapten of the first target-caged hapten-antibody conjugate complex toform a first target-unmasked hapten-antibody conjugate complex; (d)contacting the sample with first detection reagents to label the firsttarget-unmasked hapten-antibody conjugate complex or the first target;and (e) detecting the labeled first target-unmasked hapten-antibodyconjugate complex or labeled first target.

In some embodiments, the first detection reagents comprise (i) asecondary antibody specific to the unmasked hapten of the firsttarget-unmasked hapten-antibody complex, the secondary antibodyconjugated to a first enzyme such that the secondary antibody labels thefirst target-unmasked hapten-antibody complex with the first enzyme; and(ii) a first substrate for the first enzyme. In some embodiments, thefirst substrate is a chromogenic substrate or a fluorescent substrate.In some embodiments, the first detection reagents include amplificationcomponents to label the unmasked enzyme of the first target-unmaskedhapten-antibody conjugate complex with a plurality of first reportermoieties. In some embodiments, the plurality of first reporter moietiesare haptens. In some embodiments, the first detection reagents furthercomprise secondary antibodies specific to the plurality of firstreporter moieties, each secondary antibody conjugated to a secondreporter moiety. In some embodiments, the second reporter moiety isselected from the group consisting of an amplification enzyme or afluorophore. In some embodiments, the second reporter moiety is anamplification enzyme and wherein the first detection reagents furthercomprise a first chromogenic substrate or a fluorescent substrate forthe amplification enzyme. In some embodiments, the method furthercomprises contacting the sample with a second substrate specific for theunmasking enzyme of the second target-unmasking enzyme-antibodyconjugate complex and detecting signals corresponding to a product of areaction between the second substrate and the unmasking enzyme.

In another aspect of the present disclosure is a method for analyzing asample to determine whether a first target is proximal to a secondtarget, the method comprising: (a) forming a second target-unmaskingenzyme-antibody conjugate complex; (b) forming a first target-cagedhapten-antibody conjugate complex; (c) unmasking the caged hapten of thefirst target-caged hapten-antibody conjugate complex to form a firsttarget-unmasked hapten-antibody conjugate complex; (d) performing asignal amplification step to label the first target-unmaskedhapten-antibody conjugate complex with a plurality of reporter moieties;and (e) detecting the plurality of reporter moieties.

In some embodiments, the plurality of reporter moieties are haptens; andwherein the method further comprises introducing secondary antibodiesspecific to the plurality of first reporter moieties, each secondaryantibody conjugated to a second reporter moiety. In some embodiments,the second reporter moiety is an amplification enzyme and wherein themethod further comprises introducing a chromogenic substrate or afluorescent substrate for the amplification enzyme. In some embodiments,the method further comprises detecting a total amount of target in thesample.

In another aspect of the present disclosure is a method for analyzing asample to determine whether a first target is proximal to a secondtarget, the method comprising: (a) forming a second target-unmaskingenzyme-antibody conjugate complex; (b) forming a first target-cagedhapten-antibody conjugate complex; (c) performing a decaging step suchthat an unmasking enzyme of the second target-unmasking enzyme-antibodyconjugate complex reacts with an enzyme substrate portion of the firsttarget-caged hapten-antibody conjugate complex to form a firsttarget-unmasked hapten-antibody conjugate complex; (d) contacting thesample with first detection reagents to label the first target-unmaskedhapten-antibody conjugate complex or the first target; and (e) detectingthe labeled first target-unmasked hapten-antibody conjugate complex orlabeled first target.

In some embodiments, the decaging step comprises changing thetemperature of the sample. In some embodiments, the decaging stepcomprises altering a pH of the sample. In some embodiments, the decagingstep comprises introducing one or more washing steps. In someembodiments, the decaging step comprises adding cofactors for theunmasking enzyme. In some embodiments, the method further comprisesdetecting a total amount of target in the sample.

In another aspect of the present disclosure is a method for analyzing asample to determine whether a first target is proximal to a secondtarget, the method comprising: (a) contacting the sample with a cagedhapten-antibody conjugate specific to the first target to form a firsttarget-caged hapten-antibody conjugate complex; (b) contacting thesample with an unmasking enzyme-antibody conjugate specific to thesecond target to form a second target-unmasking enzyme-antibodyconjugate complex, wherein an unmasking enzyme of the unmaskingenzyme-antibody conjugate is selected such that it is capable ofreacting with an enzyme substrate portion of the caged hapten-antibodyconjugate to form a first target-unmasked hapten-antibody conjugatecomplex; (c) contacting the sample with first detection reagents tolabel the first target-unmasked hapten-antibody conjugate complex or thefirst target; and (d) detecting the labeled first target-unmaskedhapten-antibody conjugate complex or labeled first target.

In some embodiments, a caged hapten portion of the caged hapten-antibodyconjugate is derived from a hapten selected from the group consisting ofDCC, biotin, nitropyrazole, thiazolesulfonamide, benzofurazan, and2-hydroxyquinoxaline. In some embodiments, the unmasking enzyme of theunmasking enzyme-antibody conjugate is selected from the groupconsisting of alkaline phosphatase, B-glucosidase, B-Galactosidase,B-Glucuronidase, Lipase, Sulfatase, Amidase, Protease, Nitroreductase,beta-lactamase, neuraminidase, and Urease. In some embodiments, thefirst detection reagents comprise (i) a secondary antibody specific tothe unmasked hapten of the first target-unmasked hapten-antibodycomplex, the secondary antibody conjugated to a first enzyme such thatthe secondary antibody labels first target-unmasked hapten-antibodycomplex with the first enzyme; and (ii) a first chromogenic substrate orfluorescent substrate. In some embodiments, the first enzyme isdifferent than the unmasking enzyme. In some embodiments, the firstenzyme is a peroxidase. In some embodiments, the first chromogenicsubstrate is selected from the group consisting of 3,3′-diaminobenzidine(DAB), 3-amino-9-ethylcarbazole (AEC), H1RP-Silver, andtyramide-chromogens. In some embodiments, the method further comprisescontacting the sample with a second chromogenic substrate or fluorescentsubstrate specific for the unmasking enzyme of the secondtarget-unmasking enzyme-antibody conjugate complex, wherein the firstand second chromogenic substrates are different. In some embodiments,the first detection reagents include components to amplify the amount oflabel introduced to the first target-unmasked hapten-antibody conjugatecomplex. In some embodiments, the first target is one of PD-1 or PD-L1,and the second target is the other of PD-1 or PD-L1.

In another aspect of the present disclosure is a method for analyzing asample to determine whether a first target is proximal to a secondtarget, the method comprising: (a) contacting the sample with a firstdetection probe, the first detection probe comprising one of a cagedhapten-antibody conjugate or an unmasking enzyme-antibody conjugate; (b)contacting the sample with a second detection probe, the seconddetection probe comprising the other of the caged hapten-antibodyconjugate or the unmasking enzyme-antibody conjugate; (c) contacting thesample with at least first detection reagents to label a formed unmaskedhapten-antibody conjugate target complex; (d) detecting signals from thelabeled unmasked hapten-antibody conjugate target complex.

In some embodiments, the method further comprises detecting a totalamount of target within the sample. In some embodiments, the firstdetection reagents include amplification components to label theunmasked enzyme of the first target-unmasked hapten-antibody conjugatecomplex with a plurality of first reporter moieties. In someembodiments, the plurality of first reporter moieties are haptens. Insome embodiments, the first detection reagents further comprisesecondary antibodies specific to the plurality of first reportermoieties, each secondary antibody conjugated to a second reportermoiety. In some embodiments, the second reporter moiety is selected fromthe group consisting of an amplification enzyme or a fluorophore. Insome embodiments, the second reporter moiety is an amplification enzymeand wherein the first detection reagents further comprise a firstchromogenic substrate or fluorescent substrate for the amplificationenzyme. In some embodiments, the method further comprises a decagingstep.

In another aspect of the present disclosure is a method for analyzing asample to determine whether a first target is proximal to a secondtarget, the method comprising: (a) contacting the sample with a cagedhapten-antibody conjugate specific to the first target to form a firsttarget-caged hapten-antibody conjugate complex; (b) contacting thesample with an unmasking enzyme-antibody conjugate specific to thesecond target to form a second target-unmasking enzyme-antibodyconjugate complex, wherein an unmasking enzyme of the unmaskingenzyme-antibody conjugate is selected such that it is capable ofreacting with an enzyme substrate portion of the caged hapten-antibodyconjugate to form a first target-unmasked hapten-antibody conjugatecomplex; (c) contacting the sample with a first labeling conjugate thatspecifically binds to the first target-unmasked hapten-antibodyconjugate complex, wherein the first labeling conjugate comprises afirst enzyme which differs from the unmasking enzyme; (d) contacting thesample with a first signaling conjugate comprising a first latentreactive moiety and a first chromogenic or fluorescent moiety; and (e)detecting signals from the first chromogenic or fluorescent moiety, thefirst signals being indicative of proximal first and second targets.

In some embodiments, the method further comprises contacting the samplewith a second signaling conjugate comprising a second latent reactivemoiety and a second chromogenic or fluorescent moiety, wherein the firstand second chromogenic or fluorescent moieties provide differentsignals. In some embodiments, further comprises detecting signals fromthe second chromogenic moiety, the signals from the second chromogenicor fluorescent moiety being indicative of total protein.

In another aspect of the present disclosure is a caged hapten havingFormula (I):

-   -   wherein    -   Y is selected from a carbonyl-reactive group, an amine-reactive        group, or a thiol-reactive group;    -   X is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S;    -   A is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 15 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S; and    -   ‘Caging Group’ comprises an enzyme substrate and optionally a        leaving group, wherein the leaving group portion may comprise,        in some non-limiting embodiments, a substituted or unsubstituted        5-, 6-, or 7-membered aromatic or heterocyclic ring.

In some embodiments, the 5-, 6-, or 7-membered aromatic or heterocyclicring is substituted with a moiety selected from the group consisting ofa halogen, a —S-alkyl group having between 1 and 4 carbon atoms; an—O-alkyl group having between 1 and 4 carbon atoms; a —N(H)-alkyl grouphaving between 1 and 4 carbon atoms; a —N-(alkyl)₂ group having between1 and 6 carbon atoms; and a branched or unbranched, substituted orunsubstituted alkyl group having between 1 and 4 carbon atoms. In someembodiments, the caging group has the structure of Formula (II):

-   -   wherein    -   R¹ is independently selected from H, F, Cl, Br, I, —O-methyl,        —O-ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl, —O-sec-butyl,        or —O-iso-butyl; a —S-alkyl group having between 1 and 4 carbon        atoms; an —O-alkyl group having between 1 and 4 carbon atoms; a        —N(H)-alkyl group having between 1 and 4 carbon atoms; a        —N-(alkyl)₂ group having between 1 and 6 carbon atoms; an alkyl        group having between 1 and 4 carbon atoms and optionally        substituted with N or S; cyano groups; and carboxyl groups; and    -   R² is an enzyme substrate, -alkyl-enzyme substrate, or        —O-alkyl-enzyme substrate.

In some embodiments, the enzyme substrate is selected from the groupconsisting of a phosphate group, an ester group, an amide group, asulfate group, a glycoside group, a urea group, and a nitro group. Insome embodiments, the caging group has the structure of Formula (IIB):

and

-   -   wherein R² is selected from the group consisting of a phosphate        group, an ester group, an amide group, a sulfate group, a        glycoside group, a urea group, an -alkyl-phosphate group, an        —O-alkyl-phosphate group, and a nitro group. In some        embodiments, each R¹ group is different.

In some embodiments, the caging group has the structure of Formula(IIC):

and

-   -   wherein R² is selected from the group consisting of a phosphate        group, an ester group, an amide group, a sulfate group, a        glycoside group, a urea group, an -alkyl-phosphate group, an        —O-alkyl-phosphate group, and a nitro group. In some        embodiments, each R¹ group is different.

In some embodiments, the caging group has the structure of Formula(IID):

and

-   -   wherein R² is selected from the group consisting of a phosphate        group, an ester group, an amide group, a sulfate group, a        glycoside group, a urea group, an -alkyl-phosphate group, an        —O-alkyl-phosphate group, and a nitro group.

In some embodiments, each R¹ group is different. In some embodiments, Xhas the structure of Formula (IIIA):

-   -   wherein d and e are integers each independently ranging from 4        to 18; Q is a bond, O, S, or N(R^(c))(R^(d)); Ra and R^(b) are        independently H, a C₁-C₄ alkyl group, F, Cl, or N(R^(c))(R^(d));        and R^(c) and R^(d) are independently CH₃ or H. In some        embodiments, d and e are integers each independently ranging        from 1 to 24.

In some embodiments, the caged hapten is selected from the groupconsisting of:

In another aspect of the present disclosure is a conjugate of (i) aspecific binding entity, and (ii) a caged hapten, including any of thecaged haptens cited herein. In some embodiments, the caged hapten hasthe structure of Formula (I):

-   -   wherein    -   “Hapten” is a hapten;    -   Y is selected from a carbonyl-reactive group, an amine-reactive        group, or a thiol-reactive group;    -   X is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S;    -   A is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 15 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S; and    -   ‘Caging Group’ comprises an enzyme substrate and optionally a        leaving group, wherein the leaving group portion comprises a        substituted or unsubstituted 5, 6-, or 7-membered aromatic or        heterocyclic ring.

In some embodiments, the specific binding entity of the conjugate is anantibody. In some embodiments, the conjugate has the structure ofFormula (IV):

-   -   wherein    -   “Antibody” is an antibody;    -   “Hapten” is a hapten;    -   Z is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S,    -   A is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 15 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S;    -   “Caging Group” a 5-, 6-, or 7-membered aromatic or heterocyclic        ring optionally substituted with a moiety selected from the        group consisting of a halogen, a —S-alkyl group having between 1        and 4 carbon atoms; an —O-alkyl group having between 1 and 4        carbon atoms; a —N(H)-alkyl group having between 1 and 4 carbon        atoms; a —N-(alkyl)2 group having between 1 and 6 carbon atoms;        and a branched or unbranched, substituted or unsubstituted alkyl        group having between 1 and 4 carbon atoms.    -   q is 0 or 1; and    -   n is an integer ranging from 1 to 25.

In some embodiments, the caging group has the structure of Formula (II):

-   -   wherein    -   R¹ is independently selected from H, F, Cl, Br, I, —O-methyl,        —O-ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl, —O-sec-butyl,        or —O-iso-butyl; a —S-alkyl group having between 1 and 4 carbon        atoms; an —O-alkyl group having between 1 and 4 carbon atoms; a        —N(H)-alkyl group having between 1 and 4 carbon atoms; a        —N-(alkyl)₂ group having between 1 and 6 carbon atoms; an alkyl        group having between 1 and 4 carbon atoms and optionally        substituted with N or S; cyano groups; and carboxyl groups; and    -   R² is an enzyme substrate, -alkyl-enzyme substrate, or        —O-alkyl-enzyme substrate.

In some embodiments, the enzyme substrate is selected from the groupconsisting of a phosphate group, an ester group, an amide group, asulfate group, a glycoside group, a urea group, and a nitro group.

In some embodiments, the caging group has the structure of Formula(IIB):

and

-   -   wherein R² is selected from the group consisting of a phosphate        group, an ester group, an amide group, a sulfate group, a        glycoside group, a urea group, an -alkyl-phosphate group, an        —O-alkyl-phosphate group, and a nitro group. In some        embodiments, each R¹ group is different.

In some embodiments, the caging group has the structure of Formula(IIC):

and

-   -   wherein R² is selected from the group consisting of a phosphate        group, an ester group, an amide group, a sulfate group, a        glycoside group, a urea group, an -alkyl-phosphate group, an        —O-alkyl-phosphate group, and a nitro group. In some        embodiments, R¹ group is different.

In some embodiments, the caging group has the structure of Formula(IID):

and

-   -   wherein R² is selected from the group consisting of a phosphate        group, an ester group, an amide group, a sulfate group, a        glycoside group, a urea group, an -alkyl-phosphate group, an        —O-alkyl-phosphate group, and a nitro group. In some        embodiments, each R¹ group is different.

In some embodiments, the antibody is a secondary antibody. In someembodiments, the antibody is a primary antibody. In some embodiments,the primary antibody is specific for a target selected from the groupconsisting of PD-L1/CD80(B7-1); CTLA-4/CD80(B7-1); CTLA-4/CD86(B7-1);PD-L2/PD-1; any combination of a ErbB family (Her1 (EGFR), Her2, Her3,Her4); any combination of DNA mixed match repair proteins (MLH1, MLH3,MSH2, MSH3, MSH6, PMS1 and PMS2); post translational modifications(PTM)-phosphorylated proteins (combining ananti-phosphotyrosine/phosphoserine/phosphothreonine/phosphohistidineantibody with any antibody specific against the target in question); andPTM-ubiquitinated proteins.

In another aspect of the present disclosure is a method for detectingmultiple targets within a sample comprising: (a) contacting the samplewith a caged hapten-antibody conjugate specific to the first target toform a first target-caged hapten-antibody conjugate complex; (b)contacting the sample with an unmasking enzyme-antibody conjugatespecific to the second target to form a second target-unmaskingenzyme-antibody conjugate complex, wherein an unmasking enzyme of theunmasking enzyme-antibody conjugate is selected such that it is capableof reacting with an enzyme substrate portion of the cagedhapten-antibody conjugate to form a first target-unmaskedhapten-antibody conjugate complex; (c) contacting the sample with firstdetection reagents to label the first target-unmasked hapten-antibodyconjugate complex or the first target; (d) contacting the sample with afirst detection probe specific to a third target to form a thirdtarget-detection probe complex; (e) contacting the sample with seconddetection reagents to label the third target-detection probe complex;(f) detecting the labeled first target-unmasked hapten-antibodyconjugate complex or labeled first target; and (g) detecting the labeledthird target-detection probe complex. In some embodiments, the methodfurther comprises detecting total protein within the sample. In someembodiments, the first detection probe comprises an antibody. In someembodiments, the first detection probe comprises a nucleic acid probe.In some embodiments, the method further comprised contacting the samplewith a second detection probe specific to a fourth target to form afourth target-detection probe complex. In some embodiments, the methodfurther comprises inactivating the unmasking enzyme prior to contactingthe sample with second detection reagents. In some embodiments, themethod further comprises inactivating the first and second targetcomplexes prior to contacting the sample with a probe to label the thirdtarget and second detection reagents.

In another aspect of the present disclosure is a caged hapten compoundhaving Formula (IE) or (IF):

-   -   wherein    -   “Hapten” is a hapten;    -   A is a group comprising a branched or unbranched, substituted or        unsubstituted, saturated or unsaturated aliphatic group having        between 1 and 15 carbon atoms, and optionally having one or more        heteroatoms selected from the group consisting of O, N, or S;    -   Y is selected from a carbonyl-reactive group, an amine-reactive        group, or a thiol-reactive group;    -   X is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S;    -   W is a 5-, 6-, or 7-membered substituted or unsubstituted        aromatic or heterocyclic group;    -   each R¹ is independently selected from H, F, Cl, Br, I,        —O-methyl, —O— ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl,        —O-sec-butyl, or —O-iso-butyl; a —S-alkyl group having between 1        and 4 carbon atoms; an —O-alkyl group having between 1 and 4        carbon atoms; a —N(H)-alkyl group having between 1 and 4 carbon        atoms; a —N-(alkyl)₂ group having between 1 and 6 carbon atoms;        an alkyl group having between 1 and 4 carbon atoms and        optionally substituted with N or S; cyano groups; and carboxyl        groups;    -   V is a bond, a substituted or unsubstituted alkyl group having        between 1 and 4 carbon atoms, or a substituted or unsubstituted        —O-alkyl group;    -   R³ is an enzyme substrate;    -   q is 0 or 1;    -   s is 0 or an integer ranging from 1 to 4; and    -   t is 0 or 1.

In some embodiments, the enzyme substrate is selected from the groupconsisting of a phosphate group, an ester group, an amide group, asulfate group, a glycoside group, a urea group, and a nitro group. Insome embodiments, the enzyme substrate is a phosphate group.

In some embodiments, each R¹ group is different.

In some embodiments, X has the structure of Formula (IIIA):

-   -   wherein d and e are integers each independently ranging from 4        to 18; Q is a bond, O, S, or N(R^(c))(R^(d)); Ra and R^(b) are        independently H, a C₁-C₄ alkyl group, F, Cl, or N(R^(c))(R^(d));        and R^(c) and R^(d) are independently CH₃ or H. In some        embodiments, d and e are integers each independently ranging        from 1 to 24.

In some embodiments, when V is a substituted-alkyl group or asubstituted —O-alkyl group, it is substituted with one or more anelectron withdrawing groups.

In some embodiments, t is 1 and V is —CH₂—.

In some embodiments, t is 1 and V is —O—CH₂—.

In some embodiments, t is 1 and V is —C(R³)₂ where R³ is a C₁-C₄ alkylgroup.

In some embodiments, t is 1 and V is —O—C(R³)₂ where R³ is a C₁-C₄ alkylgroup.

In some embodiments, s is 2, R¹ is —O—CH₃—, t is 1 and V is —CH₂—.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided to the Office upon request and thepayment of the necessary fee.

FIG. 1A illustrates an embodiment of an unmasked hapten.

FIG. 1B illustrates an embodiment of a caged hapten.

FIG. 1C illustrates a structure of a caging group coupled to a hapten.

FIG. 2 is a schematic illustrating the interaction between an unmaskingenzyme-antibody conjugate comprising an alkaline phosphatase (bound toTarget 2) and a caged hapten-antibody conjugate (bound to Target 1),where the unmasking enzyme of the unmasking enzyme-antibody conjugatereacts with an enzyme substrate portion of the caged hapten-antibodyconjugate (by virtue of the proximity of Target 1 and Target 2 to eachother) to provide the respective unmasked hapten, which may be detected.

FIG. 3 is a schematic illustrating an unmasking enzyme-antibodyconjugate (bound to Target 2) and a caged hapten-antibody conjugate(bound to Target 1) where the two targets are not in close proximity toeach other, such that the unmasking enzyme of the unmaskingenzyme-antibody conjugate does not interact with an enzyme substrateportion of the caged hapten-antibody conjugated, and thus the cagedhapten remains masked and unable to be detected.

FIG. 4 provides a flowchart illustrating the steps of detecting proteindimers and/or total protein in a sample.

FIG. 5 is a schematic illustrating an embodiment of an IHC stainingprotocol where a single antigen is detected with a secondary antibodylabeled with cHQ.

FIG. 6A is an image of the detection of PSA on prostate tissue with asecondary antibody labeled with cHQ and treatment with AP.

FIG. 6B is an additional image of the lack of detection of PSA onprostate tissue with a secondary antibody labeled with cHQ and no APtreatment (negative control).

FIG. 6C is an additional image of the detection of PSA on prostatetissue with a secondary antibody labeled with native HQ.

FIG. 7A is an image of total protein for Beta-Catenin as measured withDAB staining.

FIG. 7B is an image of proteins with positive proximity as measured withDAB staining.

FIG. 7C is an image of total protein for E-cadherin as measured with DABstaining.

FIG. 8A is an image of total protein for EGFR as measured with DABstaining on FFPE tonsil tissue.

FIG. 8B is an image of co-localized proteins without proximity asmeasured with the absence of DAB staining on FFPE tonsil tissue.

FIG. 8C is an image of total protein for E-cadherin as measured with DABstaining on FFPE tonsil tissue.

FIG. 9A is an image of co-localized proteins with proximity as measuredwith DAB staining on FFPE breast tissue.

FIG. 9B is an additional image of co-localized proteins withoutproximity as measured by the absence of DAB staining on FFPE breasttissue.

FIG. 10A is an image of signal intensity based on the number of cagedhapten labels on the secondary antibody.

FIG. 10B is an additional image of the increase in signal intensitybased on the increased number of caged hapten labels on the secondaryantibody.

FIG. 11A is an image of proximity signal detection as measured withHRP-Silver staining.

FIG. 11B is an additional image of proximity signal detection asmeasured with RP-Purple chromogen staining.

FIG. 12 is a schematic illustrating multiplex detection of both proteins(Target 1 and Target 2) in close proximity and total protein (Target 2).

FIG. 13A is an image demonstrating detection of proximal proteins withHRP-DAB.

FIG. 13B is an image demonstrating detection of proximal proteins withHRP-Silver and total protein with AP-Yellow.

FIG. 14 is an image demonstrating detection of the proximity signalusing HRP-Purple and total protein using AP-Yellow.

FIG. 15A illustrates the structures of caged haptens and the enzymealkaline phosphatase which may react with the enzyme substrate portionof the caged hapten.

FIG. 15B illustrates the structures of caged haptens and specifies theenzyme B-Glucosidase may react with the enzyme substrate portion of thecaged hapten.

FIG. 15C illustrates the structures of caged haptens and specifies theenzyme B-Galactosidase which may react with the enzyme substrate portionof the caged hapten.

FIG. 15D illustrates the structures of caged haptens and specifies theenzyme B-Glucuronidase which may react with the enzyme substrate portionof the caged hapten.

FIG. 15E illustrates the structures of caged haptens and specifies theenzyme Lipase which may react with the enzyme substrate portion of thecaged hapten.

FIG. 15F illustrates the structures of caged haptens and specifies theenzyme Sulfatase which may react with the enzyme substrate portion ofthe caged hapten.

FIG. 15G illustrates the structures of caged haptens and specifies theenzyme Amidase/Protease which may react with the enzyme substrateportion of the caged hapten.

FIG. 15H illustrates the structures of caged haptens and specifies theenzyme Nitroreductase which may react with the enzyme substrate portionof the caged hapten.

FIG. 15I illustrates the structures of caged haptens and specifies theenzyme Urease which may react with the enzyme substrate portion of thecaged hapten.

FIG. 16 provides an image of proximity signal detection of PD-1 andPD-L1 proteins as measured with HRP-Purple chromogen staining in a FFPEnon-small cell lung cancer (NSCLC) case (Arrows=PD-L1/PD-1 proximitysignal).

FIG. 17 provides an image of proximity signal detection of PD-1 andPD-L1 proteins as measured with HRP-Purple chromogen staining and totalprotein for PD-1 with AP-Yellow in FFPE tonsil (Arrows=PD-L1/PD-1proximity signal; Dashed circles=PD-1 protein signal).

FIG. 18 provides an image of a negative control for proximity signaldetection of PD-1 and PD-L1 proteins where the PD-1 antibody was omittedin FFPE tonsil and no signal was observed.

FIG. 19 provides an image of multiplex staining with proximity signaldetection of PD-1 and PD-L1 proteins as measured with HRP-Purplechromogen staining and CD8 with AP-Yellow in FFPE tonsil(Arrows=PD-L1/PD-1 proximity signal; Dashed circles=CD8 protein signal).

FIG. 20A is an image of total protein for Her2 as measured with DABstaining.

FIG. 20B is an image of Her2/Her3 proteins with positive proximity asmeasured with DAB staining.

FIG. 20C is an image of total protein for Her3 as measured with DABstaining.

FIG. 21 is a schematic illustrating the interaction between an unmaskingenzyme-antibody conjugate comprising an alkaline phosphatase (bound tothe antibody stack on Target 2) and a caged hapten-antibody conjugate(bound to the antibody stack on Target 1), where the unmasking enzyme ofthe unmasking enzyme-antibody conjugate reacts with an enzyme substrateportion of the caged hapten-antibody conjugate (by virtue of theproximity of Target 1 and Target 2 to each other) to provide therespective unmasked hapten, which may be detected by subsequentamplification schemes.

FIG. 22A is an image of E-Cadherin and β-Catenin proteins with positiveproximity as measured with DAB staining using a caged hapten of Formulas(IC) or (ID).

FIG. 22B is an image of E-Cadherin and β-Catenin proteins with positiveproximity as measured with DAB staining using a caged hapten of Formulas(IE) or (IF) and showing the same degree of specific signal as the cagedhapten of Formulas (IC) or (ID).

FIG. 23 is a schematic illustrating a control experiment to investigatethe completeness of the caging of the haptens on the antibody conjugate.Undesired signal may be generated if the anti-hapten conjugate 209recognizes an uncaged hapten 206. When all the haptens 206 are cagedthen no signal would be generated.

FIG. 24A is an image of a control staining for Her2 protein on FFPESKBR3 cell lines using an antibody conjugate with a caged hapten ofFormulas (IC) or (ID).

Positive signal indicates the incomplete caging of the hapten in theantibody conjugate.

FIG. 24B is an image of a control staining for Lambda protein on FFPEtonsil tissue using an antibody conjugate with a caged hapten ofFormulas (IC) or (ID).

Positive signal indicates the incomplete caging of the hapten in theantibody conjugate.

FIG. 24C is an image of a control staining for Her2 protein on FFPESKBR3 cell lines using an antibody conjugate with a caged hapten ofFormulas (IE) or (IF). The absence of positive signal indicates thecomplete caging of the hapten in the antibody conjugate.

FIG. 24D is an image of a control staining for Lambda protein on FFPEtonsil tissue using an antibody conjugate with a caged hapten ofFormulas (IE) or (IF). The absence of positive signal indicates thecomplete caging of the hapten in the antibody conjugate.

FIG. 25 is a graph illustrating the stability of various caged haptens,the stability shown as the percent of decaging over time (days).

DETAILED DESCRIPTION

Disclosed herein are caged haptens and their method of synthesis. Alsodisclosed herein are conjugates comprising a caged hapten. As will bedescribed in more detail herein, the caged hapten conjugates may be usedto detect proximal antigens in tissue samples. These and otherembodiments are described herein.

Definitions

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. The term “includes” is defined inclusively, suchthat “includes A or B” means including A, B, or A and B.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of or “exactly one of,” or, when used inthe claims, “consisting of,” will refer to the inclusion of exactly oneelement of a number or list of elements. In general, the term “or” asused herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

The terms “comprising,” “including,” “having,” and the like are usedinterchangeably and have the same meaning. Similarly, “comprises,”“includes,” “has,” and the like are used interchangeably and have thesame meaning. Specifically, each of the terms is defined consistent withthe common United States patent law definition of “comprising” and istherefore interpreted to be an open term meaning “at least thefollowing,” and is also interpreted not to exclude additional features,limitations, aspects, etc. Thus, for example, “a device havingcomponents a, b, and c” means that the device includes at leastcomponents a, b and c. Similarly, the phrase: “a method involving stepsa, b, and c” means that the method includes at least steps a, b, and c.Moreover, while the steps and processes may be outlined herein in aparticular order, the skilled artisan will recognize that the orderingsteps and processes may vary.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

As used herein, alkaline phosphatase (AP) is an enzyme that removes (byhydrolysis) and transfers phosphate group organic esters by breaking thephosphate-oxygen bond, and temporarily forming an intermediateenzyme-substrate bond. For example, AP hydrolyzes naphthol phosphateesters (a substrate) to phenolic compounds and phosphates. The phenolscouple to colorless diazonium salts (chromogen) to produce insoluble,colored azo dyes.

As used herein, the term “alkyl” refers to a straight or branchedhydrocarbon chain that comprises a fully saturated (no double or triplebonds) hydrocarbon group. By way of example only, the alkyl group mayhave 1 to 20 carbon atoms (whenever it appears herein, a numerical rangesuch as “1 to 20” refers to each integer in the given range; e.g., “1 to20 carbon atoms” means that the alkyl group may consist of 1 carbonatom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20carbon atoms, although the present definition also covers the occurrenceof the term “alkyl” where no numerical range is designated). As notedfurther herein, the alkyl group of the compounds may be designated as“C₁-C₄ alkyl” or similar designations. By way of example only, “C₁-C₄alkyl” indicates that there are one to four carbon atoms in the alkylchain, i.e., the alkyl chain is selected from methyl, ethyl, propyl,iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkylgroups include, but are in no way limited to, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tertiary butyl, pentyl and hexyl. The alkylgroup may be substituted or unsubstituted, as defined herein.

As used herein, the term “antibody,” occasionally abbreviated “Ab,”refers to immunoglobulins or immunoglobulin-like molecules, including byway of example and without limitation, IgA, IgD, IgE, IgG and IgM,combinations thereof, and similar molecules produced during an immuneresponse in any vertebrate, (e.g., in mammals such as humans, goats,rabbits and mice) and antibody fragments that specifically bind to amolecule of interest (or a group of highly similar molecules ofinterest) to the substantial exclusion of binding to other molecules.Antibody further refers to a polypeptide ligand comprising at least alight chain or heavy chain immunoglobulin variable region whichspecifically recognizes and binds an epitope of an antigen. Antibodiesmay be composed of a heavy and a light chain, each of which has avariable region, termed the variable heavy (VH) region and the variablelight (VL) region. Together, the VH region and the VL region areresponsible for binding the antigen recognized by the antibody. The termantibody also includes intact immunoglobulins and the variants andportions of them well known in the art.

As used herein, the phrase “antibody conjugates,” refers to thoseantibodies conjugated (either directly or indirectly) to one or morelabels, where the antibody conjugate is specific to a particular targetand where the label is capable of being detected (directly orindirectly), such as with a secondary antibody (an anti-label antibody).For example, an antibody conjugate may be coupled to a hapten such asthrough a polymeric linker and/or spacer, and the antibody conjugate, bymeans of the hapten, may be indirectly detected. As an alternativeexample, an antibody conjugate may be coupled to a chromogen, such asthrough a polymeric linker and/or spacer, and the antibody conjugate maybe detected directly. Antibody conjugates are described further in USPublication No. 2014/0147906 and U.S. Pat. Nos. 8,658,389; 8,686,122;8,618,265; 8,846,320; and 8,445,191. By way of a further example, theterm “antibody conjugates” includes those antibodies conjugated to anenzyme, e.g. HRP or AP.

As used herein, the term “antigen” refers to a compound, composition, orsubstance that may be specifically bound by the products of specifichumoral or cellular immunity, such as an antibody molecule or T-cellreceptor. Antigens can be any type of molecule including, for example,haptens, simple intermediary metabolites, sugars (e.g.,oligosaccharides), lipids, and hormones as well as macromolecules suchas complex carbohydrates (e.g., polysaccharides), phospholipids, nucleicacids and proteins.

As used herein, the term a “biological sample” can be any solid or fluidsample obtained from, excreted by or secreted by any living organism,including without limitation, single celled organisms, such as bacteria,yeast, protozoans, and amoebas among others, multicellular organisms(such as plants or animals, including samples from a healthy orapparently healthy human subject or a human patient affected by acondition or disease to be diagnosed or investigated, such as cancer).For example, a biological sample can be a biological fluid obtainedfrom, for example, blood, plasma, serum, urine, bile, ascites, saliva,cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion,a transudate, an exudate (for example, fluid obtained from an abscess orany other site of infection or inflammation), or fluid obtained from ajoint (for example, a normal joint or a joint affected by disease). Abiological sample can also be a sample obtained from any organ or tissue(including a biopsy or autopsy specimen, such as a tumor biopsy) or caninclude a cell (whether a primary cell or cultured cell) or mediumconditioned by any cell, tissue or organ. In some examples, a biologicalsample is a nuclear extract. In certain examples, a sample is a qualitycontrol sample, such as one of the disclosed cell pellet sectionsamples. In other examples, a sample is a test sample. Samples can beprepared using any method known in the art by of one of ordinary skill.The samples can be obtained from a subject for routine screening or froma subject that is suspected of having a disorder, such as a geneticabnormality, infection, or a neoplasia. The described embodiments of thedisclosed method can also be applied to samples that do not have geneticabnormalities, diseases, disorders, etc., referred to as “normal”samples. Samples can include multiple targets that can be specificallybound by one or more detection probes.

As used herein, the term “chromophore” refers to a molecule or a part ofa molecule (e.g. a chromogenic substrate) responsible for its color.Color arises when a molecule absorbs certain wavelengths of visiblelight and transmits or reflects others. A molecule having an energydifference between two different molecular orbitals falling within therange of the visible spectrum may absorb visible light and thus be aptlycharacterized as a chromophore. Visible light incident on a chromophoremay be absorbed thus exciting an electron from a ground state molecularorbital into an excited state molecular orbital.

As used herein, the term “conjugate” refers to two or more molecules ormoieties (including macromolecules or supra-molecular molecules) thatare covalently linked into a larger construct. In some embodiments, aconjugate includes one or more biomolecules (such as peptides, proteins,enzymes, sugars, polysaccharides, lipids, glycoproteins, andlipoproteins) covalently linked to one or more other molecules moieties.

As used herein, the terms “couple” or “coupling” refers to the joining,bonding (e.g. covalent bonding), or linking of one molecule or atom toanother molecule or atom.

As used herein, the term “detectable moiety” refers to a molecule ormaterial that can produce a detectable (such as visually, electronicallyor otherwise) signal that indicates the presence (i.e. qualitativeanalysis) and/or concentration (i.e. quantitative analysis) of the labelin a sample. A detectable signal can be generated by any known or yet tobe discovered mechanism including absorption, emission and/or scatteringof a photon (including radio frequency, microwave frequency, infraredfrequency, visible frequency and ultra-violet frequency photons).Examples of detectable moieties include chromogenic, fluorescent,phosphorescent and luminescent molecules and materials.

As used herein, the term “epitopes” refers to an antigenic determinant,such as continuous or non-continuous peptide sequences on a moleculethat are antigenic, i.e. that elicit a specific immune response. Anantibody binds to a particular antigenic epitope.

As used herein, horseradish peroxidase (HRP) is an enzyme that can beconjugated to a labeled molecule. It produces a colored, fluorometric,or luminescent derivative of the labeled molecule when incubated with aproper substrate, allowing it to be detected and quantified. HRP acts inthe presence of an electron donor to first form an enzyme substratecomplex and then subsequently acts to oxidize an electronic donor. Forexample, HRP may act on 3,3′-diaminobenzidine hydrochloride (DAB) toproduce a detectable color. HRP may also act upon a labeled tyramideconjugate, or tyramide like reactive conjugates (i.e. ferulate,coumaric, caffeic, cinnamate, dopamine, etc.), to deposit a colored orfluorescent or colorless detectable moiety for tyramide signalamplification (TSA).

As used herein, the terms “multiplex,” “multiplexed,” or “multiplexing”refer to detecting multiple targets in a sample concurrently,substantially simultaneously, or sequentially. Multiplexing can includeidentifying and/or quantifying multiple distinct nucleic acids (e.g.,DNA, RNA, mRNA, miRNA) and polypeptides (e.g., proteins) bothindividually and in any and all combinations.

As used herein, the term “primary antibody” refers to an antibody whichbinds specifically to the target protein antigen in a tissue sample. Aprimary antibody is generally the first antibody used in animmunohistochemical procedure.

As used herein, the term “secondary antibody” herein refers to anantibody which binds specifically to a primary antibody, thereby forminga bridge between the primary antibody and a subsequent reagent (e.g. alabel, an enzyme, etc.), if any. The secondary antibody is generally thesecond antibody used in an immunohistochemical procedure.

As used herein, the term “specific binding entity” refers to a member ofa specific-binding pair. Specific binding pairs are pairs of moleculesthat are characterized in that they bind each other to the substantialexclusion of binding to other molecules (for example, specific bindingpairs can have a binding constant that is at least 10³ M⁻¹ greater, 10⁴M⁻¹ greater or 10⁵ M⁻¹ greater than a binding constant for either of thetwo members of the binding pair with other molecules in a biologicalsample). Particular examples of specific binding moieties includespecific binding proteins (for example, antibodies, lectins, avidinssuch as streptavidins, and protein A). Specific binding moieties canalso include the molecules (or portions thereof) that are specificallybound by such specific binding proteins.

Whenever a group or moiety is described as being “substituted” or“optionally substituted” that group may be unsubstituted or substitutedwith one or more of the indicated substituents. Likewise, when a groupis described as being “substituted or unsubstituted” if substituted, thesubstituent(s) may be selected from one or more the indicatedsubstituents. If no substituents are indicated, it is meant that theindicated “optionally substituted” or “substituted” group may besubstituted with one or more group(s) individually and independentlyselected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl,(heteroalicyclyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy,acyl, mercapto, alkylthio, arylthio, cyano, cyanate, halogen,thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl,C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, protectedC-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro,silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy,trihalomethanesulfonyl, trihalomethanesulfonamido, an amino, ether,amino (e.g. a mono-substituted amino group or a di-substituted aminogroup), and protected derivatives thereof. Any of the above groups mayinclude one or more heteroatoms, including O, N, or S. For example,where a moiety is substituted with an alkyl group, that alkyl group maycomprise a heteroatom selected from O, N, or S (e.g.—(CH₂—CH₂—O—CH₂—CH₂)—).

As used herein, the term “target” refers to any molecule for which thepresence, location and/or concentration is or can be determined.Examples of target molecules include proteins, epitopes, nucleic acidsequences, and haptens, such as haptens covalently bonded to proteins.Target molecules are typically detected using one or more conjugates ofa specific binding molecule and a detectable label.

As used herein, the terms “tyramide signal amplification” or “TSA” referto an enzyme-mediated detection method that utilizes the catalyticactivity of a peroxidase (such as horseradish peroxidase) to generatehigh-density labeling of a target molecule (such as a protein or nucleicacid sequence) in situ. TSA typically involves three basic steps: (1)binding of a specific binding member (e.g., an antibody) to the targetfollowed by secondary detection of the specific binding member with asecond peroxidase-labeled specific binding member; (2) activation ofmultiple copies of a labeled tyramide derivative (e.g., a hapten-labeledtyramide) by the peroxidase; and (3) covalent coupling of the resultinghighly reactive tyramide radicals to residues (e.g., the phenol moietyof protein tyrosine residues) proximal to the peroxidase-targetinteraction site, resulting in deposition of haptens proximally(diffusion and reactivity mediated) to the target. In some examples ofTSA, more or fewer steps are involved; for example, the TSA method canbe repeated sequentially to increase signal. Methods of performing TSAand commercial kits and reagents for performing TSA are available (see,e.g., AmpMap Detection Kit with TSA™, Cat. No. 760-121, Ventana MedicalSystems, Tucson, Ariz.; Invitrogen; TSA kit No. T-20911, InvitrogenCorp, Carlsbad, Calif.). Other enzyme-catalyzed, hapten or signalinglinked reactive species can be alternatively used as they may becomeavailable.

Caged Haptens

In one aspect of the present disclosure are “caged haptens,” such asillustrated in FIG. 1B. As those of skill in the art will appreciate,haptens are small molecules that anti-hapten antibodies have been raisedagainst. A “caged hapten” is a hapten whose structure has been modifiedsuch that a suitable anti-hapten antibody no longer recognizes themolecule and no binding event occurs. In effect, the hapten's identityis “masked” or “protected.” The caged haptens disclosed herein have beendesigned with an enzyme cleavable cage such that the respective hapten,i.e. the un-caged or unmasked hapten, is released by enzymatic treatmentto regenerate the native hapten (see, for example, FIG. 2, whichillustrates the unmasking of a caged hapten via enzymatic treatment).Thus, in the presence of an appropriate enzyme, the caged hapten isunmasked and an anti-hapten antibody is free to bind to it.

In some embodiments, the caged haptens of the present disclosure havethe structure of Formula (I):

-   -   wherein    -   X is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S;    -   A is a group comprising a branched or unbranched, substituted or        unsubstituted, saturated or unsaturated aliphatic group having        between 1 and 15 carbon atoms, and optionally having one or more        heteroatoms selected from the group consisting of O, N, or S;    -   Y is a reactive group capable of forming a covalent bond with        another group; and    -   “hapten” and “Caging Group” are as described herein; and    -   q is 0 or 1.

In some embodiments, haptens include, but are not limited to, pyrazoles(e.g. nitropyrazoles); nitrophenyl compounds; benzofurazans;triterpenes; ureas (e.g. phenyl ureas); thioureas (e.g. phenylthioureas); rotenone and rotenone derivatives; oxazole (e.g. oxazolesulfonamides); thiazoles (e.g. thiazole sulfonamides); coumarin andcoumarin derivatives; and cyclolignans. Additional non-limiting examplesof haptens include thiazoles; nitroaryls; benzofurans; triperpenes; andcyclolignans.

Specific examples of haptens include di-nitrophenyl, biotin,digoxigenin, and fluorescein, and any derivatives or analogs thereof.Other haptens are described in U.S. Pat. Nos. 8,846,320; 8,618,265;7,695,929; 8,481,270; 9,017,954; and 9,575,067 the disclosures of whichare incorporated herein by reference in their entirety.

Other non-liming examples of haptens include di-nitrophenol, biotin, anddigoxigenin. Additional haptens are disclosed within U.S. Pat. Nos.8,618,265 and 9,575,067 describe haptens including pyrazoles,particularly nitropyrazoles; nitrophenyl compounds; benzofurazans;triterpenes; ureas and thioureas, particularly phenyl ureas, and evenmore particularly phenyl thioureas; rotenone and rotenone derivatives,also referred to herein as rotenoids; oxazole and thiazoles,particularly oxazole and thiazole sulfonamides; coumarin and coumarinderivatives; cyclolignans, the disclosure of which are herebyincorporated by reference herein in their entireties. Yet additionalexamples of haptens and methods for their preparation and use aredisclosed in U.S. Pat. No. 7,695,929, which is incorporated in itsentirety herein by reference.

In some embodiments, the enzyme substrate portion is coupled to theleaving group portion, such as in the caged hapten hapten compounds ofFormula (IA). In some embodiments, the caging group is an enzymecleavable moiety comprised of two covalently bonded components, namely(i) a leaving group portion, and (ii) an enzyme substrate portion, asillustrated in Formula (IA).

-   -   wherein each of Y, X, “Hapten,” A, q, “Leaving Group,” and        “Enzyme Substrate” are as defined herein.

In some embodiments, the leaving group comprises a 5-, 6-, or 7-memberedaromatic ring or heterocyclic ring, where any position of the ring maybe substituted or unsubstituted. In some embodiments, the leaving groupis a substituted or unsubstituted 5-, 6-, or 7-membered heterocyclicring having one, two, or three heteroatoms selected from O, N, or S. Insome embodiments, the leaving group is selected from a substituted orunsubstituted furan, pyrrole, thiophene, imidazole, pyrazole, oxazole,isoxazole, thiazole, pyridine, pyrazine, pyrimidine, pyridazine, atriazine, 2H-pyran, 4H-pyran, 2H-thiopyran, 4H-thiopyran, an oxazine, ora thiazine. In some embodiments, the 5-, 6-, or 7-membered aromatic orheterocyclic ring is substituted with a halogen, a —S-alkyl group havingbetween 1 and 4 carbon atoms; an —O-alkyl group having between 1 and 4carbon atoms; a —N(H)-alkyl group having between 1 and 4 carbon atoms; a—N-(alkyl)₂ group having between 1 and 6 carbon atoms; or a branched orunbranched, substituted or unsubstituted alkyl group having between 1and 4 carbon atoms which itself may comprise one or more heteroatomsselected from O, N, or S and/or which may be substituted with one ormore halogens. In some embodiments, the leaving group may be substitutedwith a nitro group, a cyano group, or a carboxy group.

In other embodiments, the caging group comprises an enzyme substrate,coupled directly or indirectly to the hapten as in Formula (IB):

-   -   wherein each of Y, X, “Hapten,” A, q, and “Enzyme Substrate” are        as defined herein.

Examples of suitable enzyme substrates (and the enzymes that act uponthem) include, but are not limited to, phosphate groups (acted upon byan alkaline phosphatase), ester groups (acted upon by a lipase); sulfategroups (acted upon by a sulfatase); glycoside groups (acted upon by aglycosylase); amide groups (acted upon by an amidase or a protease);urea groups (acted upon by a urease); and nitro groups (acted upon by anitroreductase).

In some embodiments, the compounds of Formula (I) have the structure ofFormula (IC) or (ID):

-   -   wherein    -   “Hapten” is as defined herein;    -   Y is selected from a carbonyl-reactive group, an amine-reactive        group, or a thiol-reactive group;    -   X is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S;    -   A is a group comprising a branched or unbranched, substituted or        unsubstituted, saturated or unsaturated aliphatic group having        between 1 and 15 carbon atoms, and optionally having one or more        heteroatoms selected from the group consisting of O, N, or S;    -   W is a 5-, 6-, or 7-membered substituted or unsubstituted        aromatic or heterocyclic group;    -   each R¹ is independently selected from H, F, Cl, Br, I,        —O-methyl, —O— ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl,        —O-sec-butyl, or —O-iso-butyl; a —S-alkyl group having between 1        and 4 carbon atoms; an —O-alkyl group having between 1 and 4        carbon atoms; a —N(H)-alkyl group having between 1 and 4 carbon        atoms; a —N-(alkyl)₂ group having between 1 and 6 carbon atoms;        an alkyl group having between 1 and 4 carbon atoms and        optionally substituted with N or S; cyano groups; and carboxyl        groups;    -   R³ is an enzyme substrate;    -   q is 0 or 1; and    -   s is 0 or an integer ranging from 1 to 4; and

In some embodiments, the compounds of Formula (I) have the structure ofFormula (IE):

-   -   wherein    -   Y is selected from a carbonyl-reactive group, an amine-reactive        group, or a thiol-reactive group;    -   X is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S;    -   A is a group comprising a branched or unbranched, substituted or        unsubstituted, saturated or unsaturated aliphatic group having        between 1 and 15 carbon atoms, and optionally having one or more        heteroatoms selected from the group consisting of O, N, or S;    -   W is a 5-, 6-, or 7-membered substituted or unsubstituted        aromatic or heterocyclic group;    -   V is a bond, an optionally substituted alkyl group having        between 1 and 4 carbon atoms, or an optionally substituted        —O-alkyl group;    -   each R¹ is independently selected from H, F, Cl, Br, I,        —O-methyl, —O— ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl,        —O-sec-butyl, or —O-iso-butyl; a —S-alkyl group having between 1        and 4 carbon atoms; an —O-alkyl group having between 1 and 4        carbon atoms; a —N(H)-alkyl group having between 1 and 4 carbon        atoms; a —N-(alkyl)₂ group having between 1 and 6 carbon atoms;        an alkyl group having between 1 and 4 carbon atoms and        optionally substituted with N or S; cyano groups; and carboxyl        groups;    -   R³ comprises an enzyme substrate;    -   “Hapten” is as defined herein;    -   q is 0 or 1;    -   s is 0 or an integer ranging from 1 to 4; and    -   t is 0 or 1.

In some embodiments, t is 1 and V is —CH₂—. In other embodiments, t is 1and V is —O—CH₂—. In yet other embodiments, t is 1 and V is —C(R¹)₂where R¹ is a C₁-C₄ alkyl group. In yet further embodiments, t is 1 andV is —O—C(R¹)₂ where R¹ is a C₁-C₄ alkyl group.

In some embodiments, when V is a substituted-alkyl group or asubstituted —O-alkyl group, it is substituted with one or more electronwithdrawing groups. In some embodiments, the electron withdrawing groupis a halogen, a carbonyl group (aldehyde, ketone, ester), a cyano group,or a CF₃ group.

In some embodiments, s is 2, R¹ is —O—CH₃—, t is 1 and V is —CH₂—. Inother embodiments. In some embodiments, s is 2, R¹ is —O—CH₃—, t is 1and V is —O—CH₂—. In yet other embodiments, s is 2, R¹ is —O—CH₃, t is 1and V is —C(R¹)₂ where R¹ is a C₁-C₄ alkyl group. In yet furtherembodiments, s is 2, R¹ is —O—CH₃, t is 1 and V is —O—C(R¹)₂ where R¹ isa C₁-C₄ alkyl group.

In some embodiments, the compounds of Formula (I) have the structure of

-   -   Formula (IF):

-   -   wherein    -   Y is selected from a carbonyl-reactive group, an amine-reactive        group, or a thiol-reactive group;    -   X is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S;    -   V is a bond, a substituted or unsubstituted alkyl group having        between 1 and 4 carbon atoms, or a substituted or unsubstituted        —O-alkyl group;    -   R³ comprises an enzyme substrate;    -   “Hapten” is as defined herein;    -   q is 0 or 1; and    -   t is 0 or 1.

In some embodiments, t is 1 and V is —CH₂—. In other embodiments, t is 1and V is —O—CH₂—. In yet other embodiments, t is 1 and V is —C(R¹)₂where R¹ is a C₁-C₄ alkyl group. In yet further embodiments, t is 1 andV is —O—C(R¹)₂ where R¹ is a C₁-C₄ alkyl group.

In some embodiments, when V is a substituted-alkyl group or asubstituted —O-alkyl group, it is substituted with one or more electronwithdrawing groups. In some embodiments, the electron withdrawing groupis a halogen, a carbonyl group (aldehyde, ketone, ester), a cyano group,or a CF₃ group.

In some embodiments, the caging group has the structure of Formula (II):

-   -   wherein    -   each R¹ is independently selected from H, F, Cl, Br, I,        —O-methyl, —O— ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl,        —O-sec-butyl, or —O-iso-butyl; a —S-alkyl group having between 1        and 4 carbon atoms; an —O-alkyl group having between 1 and 4        carbon atoms; a —N(H)-alkyl group having between 1 and 4 carbon        atoms; a —N-(alkyl)₂ group having between 1 and 6 carbon atoms;        an alkyl group having between 1 and 4 carbon atoms and        optionally substituted with N or S; cyano groups; and carboxyl        groups.

In some embodiments, R² is an enzyme substrate, -alkyl-enzyme substrate,or —O-alkyl-enzyme substrate.

In some embodiments, each R¹ group is the same. In other embodiments,each R¹ group is different (i.e. each R¹ comprises a different moiety).For example, a first R¹ group may comprise a halogen while a second R¹may comprise an alkyl group.

While Formula (II) depicts a six-membered aromatic ring comprising sixcarbon atoms, the skilled artisan will appreciate that one or moreheteroatoms (e.g. O, N, or S) may be substituted for one or more of thecarbon atoms of the aromatic ring.

In some embodiments, the ability of any leaving group to leave dependson the electronics of the hapten. In some embodiments, the electronicsof certain haptens require electron donating groups to encourage leavinggroup ability. In other embodiments, the haptens require an electronwithdrawing group. For example, depending on the pKa of the functionalgroup being caged, some haptens require the addition of electrondonating groups on the leaving group of the caging group, while othersmay require electron withdrawing groups. Without wishing to be bound byany particular theory, is believed that hapten functional groups with ahigh pKa value tend to require electron donating groups. This is mostlikely because once the leaving group is expelled, the electron donatinggroups in this position are able to directly stabilize the resultingpositive charge of the leaving group through inductive or resonanceeffects. An example of this was seen in the caged-HQ, where in theabsence of electron donating groups, the leaving group would not leavewithout excessively high reaction temperature (about 100° C.). On theother hand, it is believed that for hapten functional groups with a lowpKa value, electron withdrawing groups may be required. This is mostlikely because the once the leaving group is expelled, the resultingnegative charge on the hapten functional group has such high stabilitythat a destabilizing electronic force may be necessary on the leavinggroup to help stabilize the caged hapten against hydrolysis. It isbelieved that an electron withdrawing group could destabilize thepositive charge on the leaving group, thereby making the caged haptenmore stable toward hydrolysis.

The substituents R¹ and R² may be located at any position along the ringof Formula (II) relative to the group coupling the Caging Group to thehapten. In some embodiments, R² is positioned para to the group couplingthe Caging Group to the hapten, such as illustrated in Formula (IIA):

In other embodiments of the compounds of Formula (IIA), the substituentsR¹ may independently be positioned ortho or meta to group R². In someembodiments, each R¹ group of Formula (IIA) is different, i.e. each R¹group comprises a different moiety.

In further embodiments, the Caging Group has the structure of Formula(IIB):

In some embodiments, each R¹ group of Formula (IIB) is different, i.e.each R¹ group comprises a different moiety.

In yet further embodiments, the Caging Group has the structure ofFormula (IIC):

In some embodiments, each R¹ group of Formula (IIC) is different, i.e.each R¹ group comprises a different moiety.

In yet further embodiments, the Caging Group has the structure ofFormula (IID):

In some embodiments, each R¹ group of Formula (IID) is different, i.e.each R¹ group comprises a different moiety.

Without wishing to be bound by any particular theory, it is believedthat the hydrolytic stability and leaving group ability of the caginggroup can be different when coupled to different haptens. For example,the caging group with —OMe groups installed at the meta positionrelative to the phosphate are believed to provide a good mix ofstability and reactivity on a 2-hydroxyquinoxaline (HQ) hapten, but werebelieved to be comparatively less stable than desired when installed onthe nitropyrazole (NP) hapten. This was remedied by using a caging groupwith —OMe groups installed ortho relative to the phosphate group. Again,without wishing to be bound by any particular theory, it was believedthat the O-benzyl bond formed between the HQ hapten and the caging groupwas much more stable than the N-benzyl bond formed between the NP andthe caging group. This was similarly remedied by introducing a —OMegroup ortho to the benzyl group on the HQ hapten to help “push” theelectrons through the ring and break the bond, once the phosphate wascleaved. The NP hapten was comparatively less stable, and therefore itdid not require the same “push.”

Again, and without wishing to be bound by any particular theory, it isbelieved that a caging group with no —OMe groups may require very hightemperatures to disassemble. However, a caging group with —OMe groupsortho to the phosphate may require moderate temperature to disassemble;while a caging group with the —OMe meta to the phosphate group may beable to disassemble at room temperature. Without wishing to be bound byany particular theory, it is believed that it may be possible to addstability while also increasing reactivity by adding one or twoadditional aliphatic groups to the benzyl position. It is believed thatthis may sterically protect the benzyl group from hydrolysis, while alsocreating a more stable 2° or 3° benzyl carbocation upon disassembly.

As noted herein, upon interaction of an enzyme with the enzyme substrateportion of the caging group, the caging group undergoes an electronicchange such that the Caging Group separates from the caged hapten,resulting in an un-caged hapten or un-masked hapten. FIG. 2 illustratesthe electronic changes that occur within a caged HQ hapten uponinteraction of an alkaline phosphatase enzyme with the phosphate enzymesubstrate portion of the caging group. Similar electronic changes occurin other caged hapten systems as will be appreciated by those ofordinary skill in the art.

As noted above, X may a bond; or a group comprising a branched orunbranched, substituted or unsubstituted, saturated or unsaturatedaliphatic group having between 1 and 30 carbon atoms, and optionallyhaving one or more heteroatoms selected from the group consisting of O,N, or S. In some embodiments, X may comprise carbonyl, amine, ester,ether, amide, imine, thione or thiol groups. In other embodiments, X maycomprise one or more terminal groups selected from an amine, a carbonyl,ester, ether, amide, imine, thione, or thiol.

In some embodiments, X has the structure of Formula (IIIA):

-   -   wherein d and e are integers each independently ranging from 4        to 18; Q is a bond, O, S, or N(R^(c))(R^(d)); Ra and R^(b) are        independently H, a C₁-C₄ alkyl group, F, Cl, or N(R^(c))(R^(d));        and R^(c) and R^(d) are independently CH₃ or H. In some        embodiments, d and e are integers each independently ranging        from 1 to 24.

In other embodiments, the X has the structure depicted in Formula(IIIB):

-   -   wherein d and e are integers each independently ranging from 1        to 24; Q is a bond, O, S, or N(R^(c))(R^(d)); and R^(c) and        R^(d) are independently CH₃ or H. In other embodiments, Q is O.

In yet other embodiments, the ‘Linker’ has the structure depicted inFormula (IIIC):

-   -   wherein d and e are integers each independently ranging from 1        to 24. In some embodiments, d is 2 and e ranges from 2 through        24.

In yet other embodiments, X may include one or more alkylene oxide orPEG groups (e.g. PEG2 through PEG24). A person of ordinary skill in theart will appreciate that, as the number of oxygen atoms increases, thehydrophilicity of the compound also may increase.

Without wishing to be bound by any particular theory, it is believedthat the linker length may affect the proximity signal. For example,caged haptens comprising PEG8, PEG4 and no PEG linkers were made andtested. The longer in length the linker was, the more signal we saw onour control dimer system of E-cad and B-cat. While “more signal” mayseem like it would always be useful, in this case it was not because wealso observed signal when testing on protein markers that co-localizebut are known to not form dimers (Ki67 and Bc16). We found that using noPEG linker eliminated the signal on the co-localized control systemwhile still providing some signal on the known dimer control system. Asa result, some caged hapten embodiments do not include a PEG group.

As noted herein, A is a group comprising a branched or unbranched,substituted or unsubstituted, saturated or unsaturated aliphatic grouphaving between 1 and 15 carbon atoms, and optionally having one or moreheteroatoms selected from the group consisting of O, N, or S. In someembodiments, A has the structure of Formula (IIID):

-   -   wherein d and e are integers each independently ranging from 2        to 15; Q is a bond, O, S, or N(R^(c))(R^(d)); Ra and R^(b) are        independently H, a C₁-C₄ alkyl group, F, Cl, or N(R^(c))(R^(d));        and R^(c) and R^(d) are independently CH₃ or H. In some        embodiments, d and e are integers each independently ranging        from 1 to 12.

In some embodiments, the reactive group Y is a carbonyl-reactive group.Suitable carbonyl-reactive groups include hydrazine, hydrazinederivatives, and amine. In other embodiments, the reactive group Y is anamine-reactive group. Suitable amine-reactive groups include activeesters, such as NHS or sulfo-NHS, isothiocyanates, isocyanates, acylazides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes,carbonates, aryl halides, imidoesters, anhydrides and the like. In yetembodiments, the reactive group Y is a thiol-reactive group. Suitablethiol-reactive groups include non-polymerizable Michael acceptors,haloacetyl groups (such as iodoacetyl), alkyl halides, maleimides,aziridines, acryloyl groups, vinyl sulfones, benzoquinones, aromaticgroups that can undergo nucleophilic substitution such as fluorobenzenegroups (such as tetra and pentafluorobenzene groups), and disulfidegroups such as pyridyl disulfide groups and thiols activated withEllman's reagent.

Specific examples of caged haptens are provided below, including caged7-(Diethylamino)coumarin-3-carboxylic acid (DCC), caged biotin, cagednitropyrazole, caged thiazolesulfonamide (TS), and caged benzofurazan(BF). Each of the caged haptens below comprise a substrate for analkaline phosphatase enzyme.

Other examples of caged haptens, including specific enzymes that actupon the enzyme substrate portion of the caged hapten, are provided inFIG. 15.

Non-limiting examples of caged haptens of either Formulas (IE) or (IF)include:

Synthesis of Caged Haptens

The caged happens may be synthesized according to any methods known tothose of ordinary skill in the art. For example, a caging group may beprepared starting with a substituted 4-hydroxybenzaldehyde. The hydroxylgroup of the substituted 4-hydroxybenzaldehyde may be substituted by aphosphate group, followed by reduction of the aldehyde and substitutionof the resulting benzyl alcohol with a halogen through an Appelreaction. A caging group may then be installed on the hapten of interestby reaction of the halogen-substituted caging group with the haptenunder basic conditions (if the hapten contains multiple nucleophilicgroups, it may require protection chemistry to select the desiredposition for caging). The protected phosphate group of the caging groupmay then be deprotected using trimethylsilyl bromide, followed byreaction of the caged hapten with a reactive group (i.e maleimide) tofacilitate conjugation with an antibody.

The synthesis of a “Caged HQ Hapten” (see FIG. 1B) illustrated inExample 1 and at Scheme 3. The synthesis of a “Caged NP Hapten” isillustrated below in Scheme 1.

Synthetic Materials and Methods. MS data was collected on a WatersAcquity QDa (ESI) running Empower 3 (Waters). Analytical HPLC wasperformed on a Waters Alliance e2695 using Waters XBridge columnsrunning Empower 3 (Waters). Prep HPLC was performed on a Waters 2535with Waters Sunfire columns (Prep C18 OBD 10□m 50×250 mm) runningEmpower 3 (Waters). Prep chromatography was performed on a BiotageIsolera One using Biotage KP-Sil columns. All chemicals were purchasedfrom commercial suppliers and used as received unless otherwise noted.

Compound 4. A soln. of 3,5-dimethoxy-4-hydroxybenzaldehyde (1, 1.0 eq),potassium carbonate (5.0 eq), and 2 (1.5 eq) in DMF (2 mL per mmol 1)was heated to 60° C. in an oil bath with stirring for 4 h (check HPLC toconfirm reaction was >95% complete). The reaction mixture was quenchedby addition of 1M HCl and the organic layer was separated and collected.An additional quantity of EtOAc was added and the organics wereextracted with 1M HCl, sat'd NaHCO₃, and brine. The organic layer wasdried over MgSO4 and the solvents removed under reduced pressure to givecompound 3 as a light brown viscous oil. The crude 3 was dissolved inTHF (5 mL per mmol 1) followed by addition of NaBH4 (1.5 eq). Thereaction was stirred at RT for 4 h (check HPLC to confirm reactionwas >95% complete). The reaction mixture was diluted with EtOAc followedby slow addition of 1M HCl until bubbling ceased. The organic layer wasseparated, followed by washing with 1M HCl, sat'd NaHCO₃, and brine. Theorganic layer was dried over MgSO4 and the solvents removed underreduced pressure to give a light brown viscous oil. The residue wasdissolved in CH2Cl2 (5 mL per mmol 1) followed by cooling to 0° C. in anice bath under an N2 atmosphere. SOCl2 (2.5 eq) was then slowly added,and the reaction mixture was allowed to warm to RT. The reaction mixturewas then stirred at RT for 1 h (check HPLC to confirm reaction was >95%complete), followed by quenching by addition of sat'd NaHCO₃. Anadditional quantity of CH2Cl2 was added and the organics were extractedwith 1M HCl, sat'd NaHCO₃, and brine. The organic layer was dried overMgSO4 and the solvents removed under reduced pressure to give compound 4as a light brown viscous oil. MS (ESI) m/z (M+H)+ calcd forC18H31ClO7P+425.1, found 425.2.

Compound 6. To a stirred soln. of 5-nitro-3-pyrazolecarboxylic acid (5,1.0 eq) in DMF (16 mL per mmol 5) was added TEA (1.5 eq) and DMAP (1.5eq), followed by N,N′-disuccinimidyl carbonate (1.5 eq). The formationof the NHS ester was followed by HPLC and additional 0.1 eqN,N′-disuccinimidyl carbonate was added until the reaction was complete.N-Boc-ethylenediamine (1.5 eq) was then added and the reaction mixturestirred at RT for 15 min. The reaction was followed by HPLC andadditional 0.1 eq N-boc-ethylenediamine was added until the reaction wascomplete. The reaction mixture was diluted with EtOAc (16 mL per mmol 5)followed by slow addition of 1M HCl (16 mL per mmol 5). The organiclayer was separated, followed by washing with 1M HCl, sat'd NaHCO₃, andbrine. The organic layer was dried over MgSO4 and the solvents removedunder reduced pressure to give compound 6 as a white solid. MS (ESI) m/z(M+H-Boc)+ calcd for C6H10N503+200.1, found 200.1.

Compound 7. To a soln. of compound 6 (1.0 eq), sodium carbonate (5.0eq), and tetrabutylammonium bromide (1.5 eq) in CHCl3 (2 mL per mmol 6)was added compound 4 (1.5 eq). The reaction vessel was sealed and thereaction mixture was heated to 80° C. in an oil bath with vigorousstirring for 4 h (check HPLC to confirm reaction was >95% complete). Thereaction was removed from the oil bath and was allowed to cool to RT.The reaction mixture was diluted with EtOAc followed by slow addition of1M HCl until bubbling ceased. The organic layer was separated, followedby washing with 1M HCl and brine. The organic layer was dried over MgSO4and the solvents removed under reduced pressure to give a light brownviscous oil. The crude oil was purified by flash chromatography (hex:EA)to give compound 7 as a light brown viscous oil. MS (ESI) m/z (M+H)+calcd for C29H47N5012P+688.3, found 688.5.

Compound 10. Compound 7 was dissolved in a 9:1 mixture of CH2Cl2:TFA (5mL per mmol 7) and the resulting reaction mixture was stirred at RT for30 min (check HPLC to confirm reaction was >95% complete). The reactionmixture was diluted with toluene, followed by removal of the solventsunder reduced pressure. The residue was suspended in DMF (2 mL per mmol7), followed by addition of triethylamine (5 eq) and finally3-maleimidopropionic acid NHS ester (9, 1.1 eq). The reaction vessel wassealed and the reaction mixture was vigorously stirred at rt for 4 h(check HPLC to confirm reaction completion). The reaction mixture wasthen diluted with MeOH and directly purified by prep RP-HPLC (0.05% TFAin H2O:MeCN 99:1 to 5:95 over 40 min) to give compound 10 as a lightyellow solid. MS (ESI) m/z (M+H)+ calcd for C23H28N6013P+627.1, found627.3.

Compound 11a-b. To a soln. of compound 6 (1.0 eq) and potassiumcarbonate (5.0 eq) in CHCl3 (2 mL per mmol 6) was added compound 2 (1.5eq). The reaction vessel was sealed and the reaction mixture was heatedto 60° C. in an oil bath with vigorous stirring for 4 h (check HPLC toconfirm reaction was >95% complete). The reaction was removed from theoil bath and was allowed to cool to RT. The reaction mixture was dilutedwith EtOAc followed by slow addition of 1M HCl until bubbling ceased.The organic layer was separated, followed by washing with 1M HCl andbrine. The organic layer was dried over MgSO4 and the solvents removedunder reduced pressure to give a light brown viscous oil. The crude oilwas purified by flash chromatography (hex:EA) to give a 1:1 mixture ofcompounds 11a and 11b as a light brown viscous oil. MS (ESI) m/z(M-Boc+H)+ calcd for C15H29N507P+422.2, found 422.3.

Compound 13a-b. Compounds 11a and 11b (as a 1:1 mixture from theprevious step; 1.0 eq) was dissolved in a 9:1 mixture of CH2Cl2:TFA (5mL per mmol 11a and 11b) and the resulting reaction mixture was stirredat RT for 30 min (check HPLC to confirm reaction was >95% complete). Thereaction mixture was diluted with toluene, followed by removal of thesolvents under reduced pressure. The residue was suspended in DMF (2 mLper mmol 7), followed by addition of triethylamine (5 eq) and finally3-maleimidopropionic acid NHS ester (9, 1.1 eq). The reaction vessel wassealed and the reaction mixture was vigorously stirred at rt for 4 h(check HPLC to confirm reaction completion). The reaction mixture wasthen diluted with MeOH and directly purified by prep RP-HPLC (0.05% TFAin H2O:MeCN 99:1 to 5:95 over 40 min) to give compounds 13a and 13b aslight yellow solids. MS (ESI) m/z (M+H)+ calcd for C14H18N6010P+461.1,found 461.1.

Conjugates of Caged Haptens

The present disclosure provides novel conjugates comprising a cagedhapten. In some embodiments, the caged hapten is conjugated to specificbinding entity, e.g. an antibody or nucleic acid probe. In otherembodiments, the caged hapten is conjugated to an antibody, as inFormula (IV):

-   -   where    -   “Antibody” and “Hapten” are as defined herein;    -   A is a group comprising a branched or unbranched, substituted or        unsubstituted, saturated or unsaturated aliphatic group having        between 1 and 15 carbon atoms, and optionally having one or more        heteroatoms selected from the group consisting of O, N, or S;    -   Q is 0 or 1;    -   n is an integer ranging from 1 to 25, and    -   Z is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S. In some embodiments, Z comprises a        group having the structure of any of Formulas (IIIA), (IIIB), or        (IIIC).

In some embodiments, n is an integer ranging from 1 to 8. In otherembodiments, n is an integer ranging from 1 to 6. In yet otherembodiments, n is an integer ranging from 1 to 4. In furtherembodiments, n is an integer ranging from 2 to 5. In yet furtherembodiments, n is 3. In even further embodiments, n is 4.

In some embodiments, the compounds of Formula (IV) have the structure ofany of compounds (IVA) or (IVB):

-   -   wherein    -   “Antibody” is as defined herein,    -   “Hapten” is as defined herein;    -   A is a group comprising a branched or unbranched, substituted or        unsubstituted, saturated or unsaturated aliphatic group having        between 1 and 15 carbon atoms, and optionally having one or more        heteroatoms selected from the group consisting of O, N, or S;    -   Z is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S;    -   W is a 5-, 6-, or 7-membered substituted or unsubstituted        aromatic or heterocyclic group;    -   each R¹ is independently selected from H, F, Cl, Br, I,        —O-methyl, —O— ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl,        —O-sec-butyl, or —O-iso-butyl; a —S-alkyl group having between 1        and 4 carbon atoms; an —O-alkyl group having between 1 and 4        carbon atoms; a —N(H)-alkyl group having between 1 and 4 carbon        atoms; a —N-(alkyl)₂ group having between 1 and 6 carbon atoms;        an alkyl group having between 1 and 4 carbon atoms and        optionally substituted with N or S; cyano groups; and carboxyl        groups;    -   R³ is an enzyme substrate;    -   n is an integer ranging from 1 to 25;    -   q is 0 or 1; and    -   s is 0 or an integer ranging from 1 to 4.

In some embodiments, the compounds of Formula (IV) have the structure ofFormulas (IVC) or (IVD):

-   -   wherein    -   “Antibody” is as defined herein,    -   “Hapten” is as defined herein;    -   A is a group comprising a branched or unbranched, substituted or        unsubstituted, saturated or unsaturated aliphatic group having        between 1 and 15 carbon atoms, and optionally having one or more        heteroatoms selected from the group consisting of O, N, or S;    -   W is a 5-, 6-, or 7-membered substituted or unsubstituted        aromatic or heterocyclic group;    -   Z is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S.    -   each R¹ is independently selected from H, F, Cl, Br, I,        —O-methyl, —O— ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl,        —O-sec-butyl, or —O-iso-butyl; a —S-alkyl group having between 1        and 4 carbon atoms; an —O-alkyl group having between 1 and 4        carbon atoms; a —N(H)-alkyl group having between 1 and 4 carbon        atoms; a —N-(alkyl)₂ group having between 1 and 6 carbon atoms;        an alkyl group having between 1 and 4 carbon atoms and        optionally substituted with N or S; cyano groups; and carboxyl        groups;    -   V is a bond, a substituted or unsubstituted alkyl group having        between 1 and 4 carbon atoms, or a substituted or unsubstituted        —O-alkyl group;    -   R³ is an enzyme substrate;    -   n is an integer ranging from 1 to 25, and    -   q is 0 or 1;    -   s is 0 or an integer ranging from 1 to 4; and    -   t is 0 or 1.

In some embodiments, when V is a substituted-alkyl group or asubstituted —O-alkyl group, it is substituted with one or more anelectron withdrawing groups.

In some embodiments, the caged haptens are conjugated to primaryantibodies (e.g. a caged hapten conjugated to an antibody specific forBeta-Catenin). In other embodiments, the caged haptens are conjugated tosecondary antibodies (e.g. a caged hapten conjugated to an antibodyspecific for an anti-Beta-Catenin antibody).

The caged haptens may be coupled to any portion of an antibody. Threefunctional groups of antibodies suitable for covalent modificationsinclude (i) amines (—NH2), (ii) thiol groups (—SH), and (iii)carbohydrate residues. As such, any of the caged haptens disclosedherein may be coupled to amine residues, thiol residues, andcarbohydrate residues or any combination thereof. In some embodiments,the caged haptens are coupled to Fc portions of the antibody.

Synthesis of Caged Hapten Conjugates

The conjugates of the present disclosure may be synthesized according toany means known to those of ordinary skill in the art.

In some embodiments, a caged hapten is conjugated to a thiol group of anantibody. In some embodiments, thiol groups are first introduced to theantibody by treating the antibody with a reducing agent such asdithiothreitol (DTT) or dithioerythritol (DTE). For a mild reducingagent, such as DTE or DTT, a concentration of between about 1 mM andabout 40 mM (for example, a concentration of between about 5 mM andabout 30 mM or between about 15 mM and about 25 mM) is utilized tointroduce a limited number of thiols (such as between about 2 and about6) to the antibody, while keeping the antibody intact (which can bedetermined by size-exclusion chromatography). Following treatment withthe reducing agent, an excess of a caged hapten bearing a thiol reactivegroup (e.g. a maleimide group) is introduced to form the respectivecaged hapten-antibody conjugate.

In other embodiments, a caged hapten is conjugated to a Fc portion of anantibody. In some embodiments, an Fc portion of an antibody is firstoxidized to form an aldehyde and the caged hapten is subsequentlycoupled to the oxidized Fc portion of the antibody through a reactivefunctional group on the caged hapten (e.g. with a carbonyl-reactivegroup, such as hydrazide group).

In yet other embodiments, a caged hapten is conjugated to a lysineresidue of an antibody. As illustrated in the synthetic scheme whichfollows (Scheme 2), in some embodiments, the antibody is first treatedwith an excess of Traut's reagent (2-iminothiolane hydrochloride) beforeadding an excess of an appropriately functionalized caged hapten (e.g.one bearing a thiol reactive group, such as a maleimide group).

Following synthesis, the conjugates may be purified, such as by sizeexclusion chromatography (SEC), and then characterized, such as by gelelectrophoresis and/or UV-Vis.

Detection of Caged Hapten Antibody Conjugates

In some embodiments, detection reagents are utilized to enable detectionof caged hapten conjugates, or complex of a caged hapten conjugate and atarget. In some embodiments, the detection reagents employed arespecific to the respective unmasked hapten corresponding to the cagedhapten of any caged hapten-conjugate. Thus, the terms “respectiveunmasked hapten” or “unmasked hapten” refer to caged haptens that havebeen “un-caged” with an appropriate unmasking enzyme to reveal the“native” hapten, i.e. an unmasking enzyme that is reactive with anenzyme substrate portion of the caged hapten. The steps of “un-caging”or “unmasking” are described further herein and depicted at least inFIG. 2. As will be described herein, detection reagents may also includecomponents designed to increase signal, e.g. signal amplificationcomponents or signal amplification kits.

In some embodiments, the detection reagents specific to the unmaskedhapten are secondary antibodies specific to the unmasked hapten of thecaged hapten conjugate, i.e. anti-unmasked hapten antibodies, and arethemselves conjugated to a detectable moiety. A “detectable moiety” is amolecule or material that can produce a detectable (such as visually,electronically or otherwise) signal that indicates the presence (i.e.qualitative analysis) and/or concentration (i.e. quantitative analysis)of the caged hapten-antibody conjugate and/or unmasking enzyme-antibodyconjugate in a sample. A detectable signal can be generated by any knownor yet to be discovered mechanism including absorption, emission and/orscattering of a photon (including radio frequency, microwave frequency,infrared frequency, visible frequency and ultra-violet frequencyphotons).

In some embodiments, the detectable moiety of the anti-unmasked haptenantibody includes chromogenic, fluorescent, phosphorescent andluminescent molecules and materials, catalysts (such as enzymes) thatconvert one substance into another substance to provide a detectabledifference (such as by converting a colorless substance into a coloredsubstance or vice versa, or by producing a precipitate or increasingsample turbidity), haptens that can be detected through antibody-haptenbinding interactions using additional detectably labeled antibodyconjugates, and paramagnetic and magnetic molecules or materials. Ofcourse, the detectable moieties can themselves also be detectedindirectly, e.g. if the detectable moiety is a hapten, then yet anotherantibody specific to that detectable moiety may be utilized in thedetection of the detectable moiety, as known to those of ordinary skillin the art.

In some embodiments, the anti-unmasked hapten antibody includes adetectable moiety selected from the group consisting of Cascade Blueacetyl azide; Dapoxylsulfonic acid/carboxylic acid DY-405; Alexa Fluor405 Cascade Yellow pyridyloxazole succinimidyl ester (PyMPO); PacificBlue DY-415; 7-hydroxycoumarin-3-carboxylic acid DYQ-425; 6-FAMphosphoramidite; Lucifer Yellow; Alexa Fluor 430 Dabcyl NBDchloride/fluoride; QSY 35 DY-485XL; Cy2 DY-490; Oregon Green 488 AlexaFluor 488 BODIPY 493/503 C3 DY-480XL; BODIPY FL C3 BODIPY FL C5 BODIPYFL-X DYQ-505; Oregon Green 514 DY-510XL; DY-481XL;6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein succinimidyl ester(JOE); DY-520XL; DY-521XL; BODIPY R6G C3 erythrosin isothiocyanate;5-carboxy-2′,4′,5′,7′-tetrabromosulfonefluorescein Alexa Fluor 5326-carboxy-2′,4,4′,5′7,7′-hexachlorofluorescein succinimidyl ester (HEX);BODIPY 530/550 C3 DY-530; BODIPY TMR-X DY-555; DYQ-1; DY-556; Cy3DY-547; DY-549; DY-550; Alexa Fluor 555 Alexa Fluor 546 DY-548; BODIPY558/568 C3 Rhodamine red-X QSY 7 BODIPY 564/570 C3 BODIPY 576/589 C3carboxy-X-rhodamine (ROX); Alexa Fluor 568 DY-590; BODIPY 581/591 C3DY-591; BODIPY TR-X Alexa Fluor 594 DY-594; carboxynaphthofluoresceinDY-605; DY-610; Alexa Fluor 610 DY-615; BODIPY 630/650-X erioglaucine;Alexa Fluor 633 Alexa Fluor 635 succinimidyl ester; DY-634; DY-630;DY-631; DY-632; DY-633; DYQ-2; DY-636; BODIPY 650/665-X DY-635; Cy5Alexa Fluor 647 DY-647; DY-648; DY-650; DY-654; DY-652; DY-649; DY-651;DYQ-660; DYQ-661; Alexa Fluor 660 Cy5.5 DY-677; DY-675; DY-676; DY-678;Alexa Fluor 680 DY-679; DY-680; DY-682; DY-681; DYQ-3; DYQ-700; AlexaFluor 700 DY-703; DY-701; DY-704; DY-700; DY-730; DY-731; DY-732;DY-734; DY-750; Cy7 DY-749; DYQ-4; and Cy7.5.

Fluorophores belong to several common chemical classes includingcoumarins, fluoresceins (or fluorescein derivatives and analogs),rhodamines, resorufins, luminophores and cyanines. Additional examplesof fluorescent molecules can be found in Molecular Probes Handbook AGuide to Fluorescent Probes and Labeling Technologies, Molecular Probes,Eugene, Oreg., ThermoFisher Scientific, 11^(th) Edition. In otherembodiments, the fluorophore is selected from xanthene derivatives,cyanine derivatives, squaraine derivatives, naphthalene derivatives,coumarin derivatives, oxadiazole derivatives, anthracene derivatives,pyrene derivatives, oxazine derivatives, acridine derivatives,arylmethine derivatives, and tetrapyrrole derivatives. In otherembodiments, the fluorescent moiety is selected from a CF dye (availablefrom Biotium), DRAQ and CyTRAK probes (available from BioStatus), BODIPY(available from Invitrogen), Alexa Fluor (available from Invitrogen),DyLight Fluor (e.g. DyLight 649) (available from Thermo Scientific,Pierce), Atto and Tracy (available from Sigma Aldrich), FluoProbes(available from Interchim), Abberior Dyes (available from Abberior), DYand MegaStokes Dyes (available from Dyomics), Sulfo Cy dyes (availablefrom Cyandye), HiLyte Fluor (available from AnaSpec), Seta, SeTau andSquare Dyes (available from SETA BioMedicals), Quasar and Cal Fluor dyes(available from Biosearch Technologies), SureLight Dyes (available fromAPC, RPEPerCP, Phycobilisomes) (Columbia Biosciences), and APC, APCXL,RPE, BPE (available from Phyco-Biotech, Greensea, Prozyme, Flogen).

In other embodiments, the anti-unmasked hapten antibody is conjugated toan enzyme. In these embodiments, the final proximity signal can begenerated with any enzyme conjugated to the relevant anti-unmaskedhapten antibody, with the exception of the enzyme that is used forunmasking (e.g. an unmasking enzyme of an unmasking enzyme-antibodyconjugate, described further herein). In some embodiments, suitableenzymes include, but are not limited to, horseradish peroxidase,alkaline phosphatase, acid phosphatase, glucose oxidase, neuramindase,β-galactosidase, β-glucuronidase or β-lactamase. In other embodiments,enzymes include oxidoreductases or peroxidases (e.g. HRP). In theseembodiments, the enzyme conjugated to the anti-unmasked hapten antibodycatalyzes conversion of a chromogenic substrate, a covalent hapten, acovalent fluorophore, non-covalent chromogens, and non-covalentfluorophores to a reactive moiety which labels a sample proximal to ordirectly on the target.

Particular non-limiting examples of chromogenic compounds/substratesinclude diaminobenzidine (DAB), 4-nitrophenylphospate (pNPP), fast red,bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT),BCIP/NBT, AP Orange, AP blue, tetramethylbenzidine (TMB),2,2′-azino-di-[3-ethylbenzothiazoline sulphonate] (ABTS), o-dianisidine,4-chloronaphthol (4-CN), nitrophenyl-β-D-galactopyranoside (ONPG),o-phenylenediamine (OPD), 5-bromo-4-chloro-3-indolyl-β-galactopyranoside(X-Gal), methylumbelliferyl-β-D-galactopyranoside (MU-Gal),p-nitrophenyl-α-D-galactopyranoside (PNP),5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), 3-amino-9-ethylcarbazole (AEC), fuchsin, iodonitrotetrazolium (INT), tetrazolium blue,and tetrazolium violet. DAB, which is oxidized in the presence ofperoxidase and hydrogen peroxide, results in the deposition of a brown,alcohol-insoluble precipitate at the site of enzymatic activity.

In some embodiments, the chromogenic substrates are signaling conjugateswhich comprise a latent reactive moiety and a chromogenic moiety. Insome embodiments, the latent reactive moiety of the signaling conjugateis configured to undergo catalytic activation to form a reactive speciesthat can covalently bond with the sample or to other detectioncomponents. The catalytic activation is driven by one or more enzymes(e.g., oxidoreductase enzymes and peroxidase enzymes, like horseradishperoxidase) and results in the formation of a reactive species. Thesereactive species are capable of reacting with the chromogenic moietyproximal to their generation, i.e. near the enzyme. Specific examples ofsignaling conjugates are disclosed in US Patent Publication No.2013/0260379, the disclosure of which is hereby incorporated byreference herein in its entirety.

Other substrates include those set forth in U.S. Pat. No. 5,583,001,U.S. application publication No. 2012/0171668, and PCT/EP2015/0533556,the disclosures of which are hereby incorporate by reference herein intheir entireties. Suitable chromogenic substrates or fluorescentsubstrates coupled to TSA or QM conjugates, as noted in the aboveincorporated references, includeN,N′-biscarboxypentyl-5,5′-disulfonato-indo-dicarbocyanine (Cy5),4-(dimethylamino) azobenzene-4′-sulfonamide (Dabsyl),tetramethylrhodamine (Tamra), and Rhodamine 110 (Rhodamine).

In some embodiments, the chromogenic substrates, fluorescent substrates,or signaling conjugates are selected such that peak detectablewavelengths of any chromogenic moiety do not overlap with each other andare readily detectable by a pathologist or an optical detector (e.g. ascanner). In some embodiments, the chromogenic moieties are selectedsuch that the peak wavelengths of the different chromogenic moieties areseparated by at least about 50 nm. In other embodiments, the chromogenicmoieties are selected such that the peak wavelengths of the differentchromogenic moieties are separated by at least about 70 nm. In yet otherembodiments, the chromogenic moieties are selected such that the peakwavelengths of the different chromogenic moieties are separated by atleast about 100 nm.

In yet further embodiments, the chromogenic moieties are selected suchthat the chromogenic moieties, when introduced to the tissue specimen,provide for different colors (e.g. yellow, blue, magenta). In someembodiments, the chromogenic moieties are selected such that theyprovide a good contrast between each other, e.g. a separation of colorsthat are optically recognizable. In some embodiments, the chromogenicmoieties are selected such that when placed in close proximity of eachother provide for a signal or color that is different than the signalsor colors of either of the chromogenic moieties when observed alone.

Proximity Detection Using Caged Hapten Conjugates

As will be described in more detail herein, the present disclosureenables the detection of protein dimers or proteins in close proximityto each other. Non-limiting examples of protein-protein interactionsinclude any of the Her1/2/3/4 proteins with each other; PD-1 with PD-L1;and/or PD-L2, EGFR (Her1) with any of it associated ligands (AREG,EREG).

Without wishing to be bound by any particular theory, it is believedthat the disclosed proximity assay is more general than merely measuringprotein-protein interactions. Indeed, the disclosed assay allows for themeasurement of the proximity of binding moieties. In practice, thebinding moieties (e.g. antibodies) may be directed against targets withminimal or no distance between them. Examples of this could includesignaling events like phosphorylation of proteins. In this case, if oneantibody is directed against an epitope on a protein (e.g. HER2), and asecond antibody is directed against all phospho-tyrosines, then theproximity signal would represent all the phosphorylated HER2 proteins.This type of assay is more binary (yes/no) than pairs of proteins thatinteract with each other.

In some embodiments, the assay is able to detect protein dimers orproteins having a proximity of 5000 nm or less. In other embodiments,the assay is able to detect protein dimers or proteins having aproximity of 2500 nm or less. In yet other embodiments, the assay isable to detect protein dimers or proteins having a proximity of 1000 nmor less. In further embodiments, the assay is able to detect proteindimers or proteins having a proximity of 500 nm or less.

The skilled artisan will appreciate that the caged hapten conjugates maybe used in both simplex assays (detection of protein dimers or proteinproximity) and multiplex assays (detection of protein dimers or proteinproximity and detection of total protein). “Total protein” refers to thenormal IHC visualization of any given protein, whereas a proximitysignal is the portion of this protein that is involved in a giveninteraction. For example, and in the case of a PD-1/PD-L1 assay, theproximity signal would visualize only the interaction between PD-1 andPD-L1, whereas the total protein signal would visualize all PD-1 in thesample. Expressing the score for proximity as a numerator and the scorefor total protein as a denominator could give the fraction or percentageof PD-1 that is involved in an interaction. This may be important as adiagnostic for detecting active pharmaceutical ingredients that disturbprotein-protein interaction where the expression of protein is lessimportant that the number of interacting proteins. This is believed tohold true for phosphorylation, as described above, where instead of justreceiving an arbitrary score for the phosphorylated signal, one may beable to quantify what percentage of a give protein is phosphorylated.

With reference to FIG. 4, the detection of protein dimers takes place intwo general stages. In a first stage, a sample is labeled with anantibody conjugates at step 150. In a second stage, the sample iscontacted with first detection reagents, and optionally second detectionreagents, at step 160.

In some embodiments, a sample is first contacted at step 100 with anunmasking enzyme-antibody conjugate specific for a target, to form atarget-unmasking enzyme-antibody conjugate complex. Subsequently, thesample is then contacted at step 110 with a caged hapten-antibodyconjugate, such as those of Formula (III), and specific for anothertarget, to form a target-caged hapten-antibody conjugate complex. Aswill be appreciated by the skilled artisan, an unmasking enzyme isselected that is reactive with an enzyme substrate portion of the cagedhapten-antibody conjugate. The skilled artisan will also appreciate thatsteps 100 and 110 may be performed in any order or may be performedsimultaneously. In some embodiments, a reversible enzyme inhibitor isalso introduced prior to or simultaneously with the introduction of thecaged hapten-antibody conjugate. For example, in some embodiments, thecaged hapten-antibody conjugate may be formulated with a phosphatebuffer.

As noted herein, the caged hapten portion of the caged hapten-antibodyconjugate is capable of becoming unmasked to provide the respectiveunmasked hapten, i.e. the native hapten. As illustrated in FIG. 2, if afirst target 101 is in sufficient proximity to a second target 102, thecaged hapten-antibody conjugate 103A will be provided in proximity (theproximity being labeled 105) to the unmasking enzyme-antibody conjugate104 such that the unmasking enzyme of the unmasking enzyme-antibodyconjugate 104 may react with the enzyme substrate of the cagedhapten-antibody conjugate 103A. This in turn results in the formation ofa first target unmasked hapten-antibody conjugate complex (103B). Asillustrated in FIG. 2, the first target unmasked hapten-antibodyconjugate complex (103B) is able to bind or be recognized by otherspecific binding entities (e.g. a secondary antibody 106).

In some embodiments, the method also includes one or more “decagingsteps” where on-slide conditions are changed to enhance enzyme activity.During the antibody conjugate binding portion of the assay, when theconjugates are in excess, steps are taken to prevent incidental“decaging” of the caged hapten which would lead to false positiveresults. These steps include adding reversible enzyme inhibitors (e.g.to prevent the action of the enzyme on the caging group). For example,in the context of alkaline phosphatase (AP), these inhibitors caninclude phosphate, phenylalanine and EDTA which are believed to be ableto reduce the enzyme activity by different mechanisms. In someembodiments, non-bound antibody conjugates are removed by washing, it isthen necessary to change the on-slide conditions to be favorable for theoptimal enzyme activity to allow “decaging” to occur. Each decagingenzyme will have its own optimal conditions (buffers, salts, cofactors,temperature). These “decaging steps” including any of washing steps,steps to change the pH of the solutions or reagents present on theslide, the addition of cofactors, or the changing of temperature (e.g.temperatures ranging from about 37° C. to about 50° C.) are chosen toenhance the activity of the enzyme and promote “decaging,” withoutinterfering with the specific binding of the antibody conjugates. Forexample, and in the case of AP, washes are used to remove residualinhibitors (e.g. phosphate), buffers are also used to increase theactivity (e.g. Tris, to adjust the pH to greater than about 8), andcofactors (e.g. magnesium) are added. In some embodiments, each of theseconditions are optimized in the context of the entire assay, for examplethe activity of AP is enhanced by the addition of magnesium ions rangingin concentration from 1 mM to 1 M. However, at concentrations ofmagnesium ions greater than 100 mM the antibody binding is affected, sothis limits the amount that may be added. Another example of anoptimizable step is the temperature of the “decaging” steps. The“decaging” event after the enzymatic step is driven by thermodynamicsand can be accelerated by heating. However, temperatures greater than60° C. can negatively affect both the enzyme activity and the antibodyconjugate binding.

On the other hand, and as illustrated in FIG. 3, if a first target 101is not in sufficient proximity to a second target 102, the cagedhapten-antibody conjugate 103A will not be provided in proximity (theproximity being labeled 108) to the unmasking enzyme-antibody conjugate104. In this instance, the unmasking enzyme will not be reactive withthe enzyme substrate of the caged hapten-antibody conjugate 103A, andthus the caged hapten will remain in a masked or protected state, i.e.it is not capable of binding or being recognized by other specificbinding entities.

Referring to FIGS. 2, 4, and 12, in some embodiments, the sample is thencontacted at step 120 with first detection reagents (106), the firstdetection reagents being specific to the unmasked hapten of the firsttarget unmasked hapten-antibody conjugate complex (103B). In someembodiments, the first detection reagents include a secondary antibody(106) specific for the unmasked hapten (103B), namely an anti-unmaskedhapten antibody. In some embodiments, the anti-unmasked hapten antibody(106) is conjugated to a detectable moiety (e.g. in FIGS. 2 and 12, thedetectable moiety is a HRP enzyme, where the HRP enzyme acts upon asubstrate, such as a silver chromogenic substrate (111)). The skilledartisan will, of course, appreciate that the first detection reagents(106) will only bind if the native or unmasked hapten (103B) of thefirst target unmasked hapten-antibody conjugate complex is revealed bythe unmasking enzyme of the unmasking enzyme-antibody conjugate (104).Thus, signal (107) from the detectable moiety of the first detectionreagents (106) will only be able to be detected at step 140 if the firstand second targets (101 and 102), and, hence, the antibody conjugates(103A and 104), are in close proximity to each other. Here, detectedsignal (107) is representative of a protein dimer or proteins/targets inclose proximity.

In some embodiments, an amplification step may be carried out toincrease detectable signal. For example, amplification components may beintroduced to further label the unmasked hapten of the first targetunmasked hapten-antibody conjugate with additional reporter moieties,e.g. additional haptens or other “detectable moieties.” By way ofexample, an anti-unmasked hapten antibody conjugated to an amplificationhapten (or, in other embodiments, conjugated to an enzyme) may beintroduced to label the unmasked hapten of the first target unmaskedhapten-antibody conjugate with a plurality of amplification haptens.

Subsequently, anti-amplification hapten antibodies, each conjugated to adetectable moiety, may be introduced. In some embodiments, theanti-amplification hapten antibodies are conjugated to an enzyme, wherethe enzyme acts upon an introduced substrate to produce a signal (e.g. achromogenic substrate or a fluorescent substrate to produce a visualsignal). TSA and QM conjugates, each described herein, may be used inany amplification step. In some examples, signal amplification iscarried out using OPTIVIEW Amplification Kit (Ventana Medical Systems,Inc., Tucson, Ariz., Catalog No. 760-099).

The skilled artisan will appreciate that the unmasking enzyme of theunmasking enzyme-antibody conjugate may serve two functions, namely (i)to unmask or reveal a caged hapten; and (ii) to react with anothersubstrate (e.g. a chromogenic substrate or a fluorescent substrate) suchthat a signal independent from that generated by the unmasked hapten(i.e. the unmasked hapten-antibody conjugate complex) may be detected.Accordingly, the presently disclosed system allows for the proximitybetween two proteins to be visualized within the context of the totalprotein stain for one of the proteins. Without wishing to be bound byany particular theory, it is believed that the ability to multiplexproximity detection within the context of another protein stain is afeature that allows for the possibility of having a speedy, guided slideread (i.e. only looking for proximity signal within the total protein)or the ability to quantitate the percentage of protein that isinteracting with another (a method of scoring the proximity assay).

Referring again to FIGS. 2, 4, and 12, following the introduction of thefirst detection reagents (106), second detection reagents including asecond detectable moiety (109) may optionally be introduced to thesample at step 130 such that total protein may be detected. In someembodiments, the second detectable moiety provides signals (112)different from that of the first detectable moiety (107). In someembodiments, the second detectable moiety comprises a substrate for theunmasking enzyme, e.g. a chromogenic substrate that provides yellowsignals (109). In other embodiments, the second detectable moietycomprises a signaling conjugate. The skilled artisan will alsoappreciate that steps 120 and 130 may be performed in any order or maybe performed simultaneously.

In some embodiments, the biological samples are pre-treated with anenzyme inactivation composition to substantially or completelyinactivate endogenous peroxidase activity. For example, some cells ortissues contain endogenous peroxidase. Using an HRP conjugated antibodymay result in high, non-specific background staining. This non-specificbackground can be reduced by pre-treatment of the sample with an enzymeinactivation composition as disclosed herein. In some embodiments, thesamples are pre-treated with hydrogen peroxide only (about 1% to about3% by weight of an appropriate pre-treatment solution) to reduceendogenous peroxidase activity. Once the endogenous peroxidase activityhas been reduced or inactivated, detection kits may be added, followedby inactivation of the enzymes present in the detection kits, asprovided above. The disclosed enzyme inactivation composition andmethods can also be used as a method to inactivate endogenous enzymeperoxidase activity.

In some embodiments if the specimen is a sample embedded in paraffin,the sample can be deparaffinized using appropriate deparaffinizingfluid(s). After a waste remover removes the deparaffinizing fluid(s),any number of substances can be successively applied to the specimen.The substances can be for pretreatment (e.g., protein-crosslinking,expose nucleic acids, etc.), denaturation, hybridization, washing (e.g.,stringency wash), detection (e.g., link a visual or marker molecule to aprobe), amplifying (e.g., amplifying proteins, genes, etc.),counterstaining, coverslipping, or the like.

Automation

The assays and methods of the present disclosure may be automated andmay be combined with a specimen processing apparatus. The specimenprocessing apparatus can be an automated apparatus, such as theBENCHMARK XT instrument and SYMPHONY instrument sold by Ventana MedicalSystems, Inc. Ventana Medical Systems, Inc. is the assignee of a numberof United States patents disclosing systems and methods for performingautomated analyses, including U.S. Pat. Nos. 5,650,327, 5,654,200,6,296,809, 6,352,861, 6,827,901 and 6,943,029, and U.S. Published PatentApplication Nos. 20030211630 and 20040052685, each of which isincorporated herein by reference in its entirety. Alternatively,specimens can be manually processed.

The specimen processing apparatus can apply fixatives to the specimen.Fixatives can include cross-linking agents (such as aldehydes, e.g.,formaldehyde, paraformaldehyde, and glutaraldehyde, as well asnon-aldehyde cross-linking agents), oxidizing agents (e.g., metallicions and complexes, such as osmium tetroxide and chromic acid),protein-denaturing agents (e.g., acetic acid, methanol, and ethanol),fixatives of unknown mechanism (e.g., mercuric chloride, acetone, andpicric acid), combination reagents (e.g., Carnoy's fixative, methacarn,Bouin's fluid, B5 fixative, Rossman's fluid, and Gendre's fluid),microwaves, and miscellaneous fixatives (e.g., excluded volume fixationand vapor fixation).

If the specimen is a sample embedded in paraffin, the sample can bedeparaffinized with the specimen processing apparatus using appropriatedeparaffinizing fluid(s). After the waste remover removes thedeparaffinizing fluid(s), any number of substances can be successivelyapplied to the specimen. The substances can be for pretreatment (e.g.,protein-crosslinking, expose nucleic acids, etc.), denaturation,hybridization, washing (e.g., stringency wash), detection (e.g., link avisual or marker molecule to a probe), amplifying (e.g., amplifyingproteins, genes, etc.), counterstaining, coverslipping, or the like.

The specimen processing apparatus can apply a wide range of substancesto the specimen. The substances include, without limitation, stains,probes, reagents, rinses, and/or conditioners. The substances can befluids (e.g., gases, liquids, or gas/liquid mixtures), or the like. Thefluids can be solvents (e.g., polar solvents, non-polar solvents, etc.),solutions (e.g., aqueous solutions or other types of solutions), or thelike. Reagents can include, without limitation, stains, wetting agents,antibodies (e.g., monoclonal antibodies, polyclonal antibodies, etc.),antigen recovering fluids (e.g., aqueous- or non-aqueous-based antigenretrieval solutions, antigen recovering buffers, etc.), or the like.Probes can be an isolated nucleic acid or an isolated syntheticoligonucleotide, attached to a detectable label. Labels can includeradioactive isotopes, enzyme substrates, co-factors, ligands,chemiluminescent or fluorescent agents, haptens, and enzymes.

After the specimens are processed, a user can transport specimen-bearingslides to the imaging apparatus. The imaging apparatus used here is abrightfield imager slide scanner. One brightfield imager is the iScanCoreo™ brightfield scanner sold by Ventana Medical Systems, Inc. Inautomated embodiments, the imaging apparatus is a digital pathologydevice as disclosed in International Patent Application No.:PCT/US2010/002772 (Patent Publication No.: WO/2011/049608) entitledIMAGING SYSTEM AND TECHNIQUES or disclosed in U.S. Patent ApplicationPublication No. 2014/0178169, filed on Feb. 3, 2014, entitled IMAGINGSYSTEMS, CASSETTES, AND METHODS OF USING THE SAME. International PatentApplication No. PCT/US2010/002772 and U.S. Patent ApplicationPublication No. 2014/0178169 are incorporated by reference in theirentities. In other embodiments, the imaging apparatus includes a digitalcamera coupled to a microscope.

Counterstaining

Counterstaining is a method of post-treating the samples after they havealready been stained with agents to detect one or more targets, suchthat their structures can be more readily visualized under a microscope.For example, a counterstain is optionally used prior to coverslipping torender the immunohistochemical stain more distinct. Counterstains differin color from a primary stain. Numerous counterstains are well known,such as hematoxylin, eosin, methyl green, methylene blue, Giemsa, Alcianblue, and Nuclear Fast Red. DAPI (4′,6-diamidino-2-phenylindole) is afluorescent stain that may be used.

In some examples, more than one stain can be mixed together to producethe counterstain. This provides flexibility and the ability to choosestains. For example, a first stain, can be selected for the mixture thathas a particular attribute, but yet does not have a different desiredattribute. A second stain can be added to the mixture that displays themissing desired attribute. For example, toluidine blue, DAPI, andpontamine sky blue can be mixed together to form a counterstain.

Detection and/or Imaging

Certain aspects, or all, of the disclosed embodiments can be automated,and facilitated by computer analysis and/or image analysis system. Insome applications, precise color or fluorescence ratios are measured. Insome embodiments, light microscopy is utilized for image analysis.Certain disclosed embodiments involve acquiring digital images. This canbe done by coupling a digital camera to a microscope. Digital imagesobtained of stained samples are analyzed using image analysis software.Color or fluorescence can be measured in several different ways. Forexample, color can be measured as red, blue, and green values; hue,saturation, and intensity values; and/or by measuring a specificwavelength or range of wavelengths using a spectral imaging camera. Thesamples also can be evaluated qualitatively and semi-quantitatively.Qualitative assessment includes assessing the staining intensity,identifying the positively-staining cells and the intracellularcompartments involved in staining, and evaluating the overall sample orslide quality. Separate evaluations are performed on the test samplesand this analysis can include a comparison to known average values todetermine if the samples represent an abnormal state.

Kits

In some embodiments, the caged hapten conjugates of the presentdisclosure may be utilized as part of a “detection kit.” In general, anydetection kit may include one or more caged hapten conjugates anddetection reagents for detecting the one or more caged haptenconjugates. In some embodiments, the kit comprises a caged haptenconjugate of any of Formulas (IVC) or (IVD).

The detection kits may include a first composition comprising a cagedhapten conjugate and a second composition comprising detection reagentsspecific to the first composition, such that the caged hapten conjugatemay be detected via the detection kit. In some embodiments, thedetection kit includes a plurality of caged hapten conjugates (such asmixed together in a buffer), where the detection kit also includesdetection reagents specific for each of the plurality of caged haptenconjugates.

Of course, any kit may include other agents, including buffers;counterstaining agents; enzyme inactivation compositions;deparafinization solutions, etc. as needed for manual or automatedtarget detection. The kit may also include instructions for using any ofthe components of the kit, including methods of applying the kitcomponents to a tissue sample to effect detection of one or more targetstherein.

Samples and Targets

Samples include biological components and generally are suspected ofincluding one or more target molecules of interest. Target molecules canbe on the surface of cells and the cells can be in a suspension, or in atissue section. Target molecules can also be intracellular and detectedupon cell lysis or penetration of the cell by a probe. One of ordinaryskill in the art will appreciate that the method of detecting targetmolecules in a sample will vary depending upon the type of sample andprobe being used. Methods of collecting and preparing samples are knownin the art.

Samples for use in the embodiments of the method and with thecomposition disclosed herein, such as a tissue or other biologicalsample, can be prepared using any method known in the art by of one ofordinary skill. The samples can be obtained from a subject for routinescreening or from a subject that is suspected of having a disorder, suchas a genetic abnormality, infection, or a neoplasia. The describedembodiments of the disclosed method can also be applied to samples thatdo not have genetic abnormalities, diseases, disorders, etc., referredto as “normal” samples. Such normal samples are useful, among otherthings, as controls for comparison to other samples. The samples can beanalyzed for many different purposes. For example, the samples can beused in a scientific study or for the diagnosis of a suspected malady,or as prognostic indicators for treatment success, survival, etc.

Samples can include multiple targets that can be specifically bound by aprobe or reporter molecule. The targets can be nucleic acid sequences orproteins. Throughout this disclosure when reference is made to a targetprotein it is understood that the nucleic acid sequences associated withthat protein can also be used as a target. In some examples, the targetis a protein or nucleic acid molecule from a pathogen, such as a virus,bacteria, or intracellular parasite, such as from a viral genome. Forexample, a target protein may be produced from a target nucleic acidsequence associated with (e.g., correlated with, causally implicated in,etc.) a disease.

A target nucleic acid sequence can vary substantially in size. Withoutlimitation, the nucleic acid sequence can have a variable number ofnucleic acid residues. For example, a target nucleic acid sequence canhave at least about 10 nucleic acid residues, or at least about 20, 30,50, 100, 150, 500, 1000 residues. Similarly, a target polypeptide canvary substantially in size. Without limitation, the target polypeptidewill include at least one epitope that binds to a peptide specificantibody, or fragment thereof. In some embodiments that polypeptide caninclude at least two epitopes that bind to a peptide specific antibody,or fragment thereof.

In specific, non-limiting examples, a target protein is produced by atarget nucleic acid sequence (e.g., genomic target nucleic acidsequence) associated with a neoplasm (for example, a cancer). Numerouschromosome abnormalities (including translocations and otherrearrangements, amplification or deletion) have been identified inneoplastic cells, especially in cancer cells, such as B cell and T cellleukemias, lymphomas, breast cancer, colon cancer, neurological cancersand the like. Therefore, in some examples, at least a portion of thetarget molecule is produced by a nucleic acid sequence (e.g., genomictarget nucleic acid sequence) amplified or deleted in at least a subsetof cells in a sample.

Oncogenes are known to be responsible for several human malignancies.For example, chromosomal rearrangements involving the SYT gene locatedin the breakpoint region of chromosome 18q11.2 are common among synovialsarcoma soft tissue tumors. The t(18q11.2) translocation can beidentified, for example, using probes with different labels: the firstprobe includes FPC nucleic acid molecules generated from a targetnucleic acid sequence that extends distally from the SYT gene, and thesecond probe includes FPC nucleic acid generated from a target nucleicacid sequence that extends 3′ or proximal to the SYT gene. When probescorresponding to these target nucleic acid sequences (e.g., genomictarget nucleic acid sequences) are used in an in situ hybridizationprocedure, normal cells, which lack a t(18q11.2) in the SYT gene region,exhibit two fusions (generated by the two labels in close proximity)signals, reflecting the two intact copies of SYT. Abnormal cells with at(18q11.2) exhibit a single fusion signal.

In other examples, a target protein produced from a nucleic acidsequence (e.g., genomic target nucleic acid sequence) is selected thatis a tumor suppressor gene that is deleted (lost) in malignant cells.For example, the p16 region (including D9S1749, D9S1747, p16(INK4A),p14(ARF), D9S1748, p15(INK4B), and D9S1752) located on chromosome 9p21is deleted in certain bladder cancers. Chromosomal deletions involvingthe distal region of the short arm of chromosome 1 (that encompasses,for example, SHGC57243, TP73, EGFL3, ABL2, ANGPTL1, and SHGC-1322), andthe pericentromeric region (e.g., 19p13-19q13) of chromosome 19 (thatencompasses, for example, MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2, andGLTSCR1) are characteristic molecular features of certain types of solidtumors of the central nervous system.

The aforementioned examples are provided solely for purpose ofillustration and are not intended to be limiting. Numerous othercytogenetic abnormalities that correlate with neoplastic transformationand/or growth are known to those of ordinary skill in the art. Targetproteins that are produced by nucleic acid sequences (e.g., genomictarget nucleic acid sequences), which have been correlated withneoplastic transformation and which are useful in the disclosed methods,also include the EGFR gene (7p12; e.g., GENBANK™ Accession No. NC000007, nucleotides 55054219-55242525), the C-MYC gene (8q24.21; e.g.,GENBANK™ Accession No. NC-000008, nucleotides 128817498-128822856),D5S271 (5p15.2), lipoprotein lipase (LPL) gene (8p22; e.g., GENBANK™Accession No. NC-000008, nucleotides 19841058-19869049), RB1 (13q14;e.g., GENBANK™ Accession No. NC-000013, nucleotides 47775912-47954023),p⁵³ (17p13.1; e.g., GENBANK™ Accession No. NC-000017, complement,nucleotides 7512464-7531642)), N-MYC (2p24; e.g., GENBANK™ Accession No.NC-000002, complement, nucleotides 151835231-151854620), CHOP (12q13;e.g., GENBANK™ Accession No. NC-000012, complement, nucleotides56196638-56200567), FUS (16p11.2; e.g., GENBANK™ Accession No.NC-000016, nucleotides 31098954-31110601), FKHR (13p14; e.g., GENBANK™Accession No. NC-000013, complement, nucleotides 40027817-40138734), aswell as, for example: ALK (2p23; e.g., GENBANK™ Accession No. NC-000002,complement, nucleotides 29269144-29997936), Ig heavy chain, CCND1(11q13; e.g., GENBANK™ Accession No. NC-000011, nucleotides69165054.69178423), BCL2 (18q21.3; e.g., GENBANK™ Accession No.NC-000018, complement, nucleotides 58941559-59137593), BCL6 (3q27; e.g.,GENBANK™ Accession No. NC-000003, complement, nucleotides188921859-188946169), MALF1, AP1 (1p32-p31; e.g., GENBANK™ Accession No.NC 000001, complement, nucleotides 59019051-59022373), TOP2A (17q21-q22;e.g., GENBANK™ Accession No. NC-000017, complement, nucleotides35798321-35827695), TMPRSS (21q22.3; e.g., GENBANK™ Accession No.NC-000021, complement, nucleotides 41758351-41801948), ERG (21q22.3;e.g., GENBANK™ Accession No. NC-000021, complement, nucleotides38675671-38955488); ETV1 (7p21.3; e.g., GENBANK™ Accession No.NC-000007, complement, nucleotides 13897379-13995289), EWS (22q12.2;e.g., GENBANK™ Accession No. NC 000022, nucleotides 27994271-28026505);FLI1 (11q24.1-q24.3; e.g., GENBANK™ Accession No. NC-000011, nucleotides128069199-128187521), PAX3 (2q35-q37; e.g., GENBANK™ Accession No.NC-000002, complement, nucleotides 222772851-222871944), PAX7(1p36.2-p36.12; e.g., GENBANK™ Accession No. NC-000001, nucleotides18830087-18935219), PTEN (10q23.3; e.g., GENBANK™ Accession No.NC-000010, nucleotides 89613175-89716382), AKT2 (19q13.1-q13.2; e.g.,GENBANK™ Accession No. NC 000019, complement, nucleotides45431556-45483036), MYCL1 (1p34.2; e.g., GENBANK™ Accession No.NC-000001, complement, nucleotides 40133685-40140274), REL (2p13-p12;e.g., GENBANK™ Accession No. NC 000002, nucleotides 60962256-61003682)and CSF1R (5q33-q35; e.g., GENBANK™ Accession No. NC-000005, complement,nucleotides 149413051-149473128).

In other examples, a target protein is selected from a virus or othermicroorganism associated with a disease or condition. Detection of thevirus- or microorganism-derived target nucleic acid sequence (e.g.,genomic target nucleic acid sequence) in a cell or tissue sample isindicative of the presence of the organism. For example, the targetpeptide, polypeptide or protein can be selected from the genome of anoncogenic or pathogenic virus, a bacterium or an intracellular parasite(such as Plasmodium falciparum and other Plasmodium species, Leishmania(sp.), Cryptosporidium parvum, Entamoeba histolytica, and Giardialamblia, as well as Toxoplasma, Eimeria, Theileria, and Babesiaspecies).

In some examples, the target protein is produced from a nucleic acidsequence (e.g., genomic target nucleic acid sequence) from a viralgenome. Exemplary viruses and corresponding genomic sequences (GENBANK™RefSeq Accession No. in parentheses) include human adenovirus A(NC-001460), human adenovirus B (NC-004001), human adenovirusC(NC-001405), human adenovirus D (NC-002067), human adenovirus E(NC-003266), human adenovirus F (NC-001454), human astrovirus(NC-001943), human BK polyomavirus (V01109; GI:60851) human bocavirus(NC-007455), human coronavirus 229E (NC-002645), human coronavirus HKU1(NC-006577), human coronavirus NL63 (NC-005831), human coronavirus OC43(NC 005147), human enterovirus A (NC-001612), human enterovirus B(NC-001472), human enterovirus C(NC-001428), human enterovirus D(NC-001430), human erythrovirus V9 (NC-004295), human foamy virus(NC-001736), human herpesvirus 1 (Herpes simplex virus type 1)(NC-001806), human herpesvirus 2 (Herpes simplex virus type 2)(NC-001798), human herpesvirus 3 (Varicella zoster virus) (NC-001348),human herpesvirus 4 type 1 (Epstein-Barr virus type 1) (NC-007605),human herpesvirus 4 type 2 (Epstein-Barr virus type 2) (NC 009334),human herpesvirus 5 strain AD 169 (NC-001347), human herpesvirus 5strain Merlin Strain (NC-006273), human herpesvirus 6A (NC-001664),human herpesvirus 6B (NC-000898), human herpesvirus 7 (NC-001716), humanherpesvirus 8 type M (NC-003409), human herpesvirus 8 type P(NC-009333), human immunodeficiency virus 1 (NC-001802), humanimmunodeficiency virus 2 (NC-001722), human metapneumovirus (NC-004148),human papillomavirus-1 (NC-001356), human papillomavirus-18 (NC-001357),human papillomavirus-2 (NC-001352), human papillomavirus-54 (NC-001676),human papillomavirus-61 (NC-001694), human papillomavirus-cand90(NC-004104), human papillomavirus RTRX7 (NC-004761), humanpapillomavirus type 10 (NC 001576), human papillomavirus type 101(NC-008189), human papillomavirus type 103 (NC-008188), humanpapillomavirus type 107 (NC-009239), human papillomavirus type 16(NC-001526), human papillomavirus type 24 (NC 001683), humanpapillomavirus type 26 (NC-001583), human papillomavirus type 32(NC-001586), human papillomavirus type 34 (NC-001587), humanpapillomavirus type 4 (NC-001457), human papillomavirus type 41 (NC001354), human papillomavirus type 48 (NC-001690), human papillomavirustype 49 (NC-001591), human papillomavirus type 5 (NC-001531), humanpapillomavirus type 50 (NC-001691), human papillomavirus type 53 (NC001593), human papillomavirus type 60 (NC-001693), human papillomavirustype 63 (NC-001458), human papillomavirus type 6b (NC-001355), humanpapillomavirus type 7 (NC-001595), human papillomavirus type 71 (NC002644), human papillomavirus type 9 (NC-001596), human papillomavirustype 92 (NC-004500), human papillomavirus type 96 (NC-005134), humanparainfluenza virus 1 (NC-003461), human parainfluenza virus 2(NC-003443), human parainfluenza virus 3 (NC-001796), human parechovirus(NC-001897), human parvovirus 4 (NC-007018), human parvovirus B19(NC-000883), human respiratory syncytial virus (NC-001781), humanrhinovirus A (NC-001617), human rhinovirus B (NC-001490), humanspumaretrovirus (NC-001795), human T-lymphotropic virus 1 (NC-001436),human T-lymphotropic virus 2 (NC 001488).

In certain examples, the target protein is produced from a nucleic acidsequence (e.g., genomic target nucleic acid sequence) from an oncogenicvirus, such as Epstein-Barr Virus (EBV) or a Human Papilloma Virus (HPV,e.g., HPV16, HPV18). In other examples, the target protein produced froma nucleic acid sequence (e.g., genomic target nucleic acid sequence) isfrom a pathogenic virus, such as a Respiratory Syncytial Virus, aHepatitis Virus (e.g., Hepatitis C Virus), a Coronavirus (e.g., SARSvirus), an Adenovirus, a Polyomavirus, a Cytomegalovirus (CMV), or aHerpes Simplex Virus (HSV).

EXAMPLES Example 1—Synthesis of[4-({[3-({2-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido]ethyl}carbamoyl)quinoxalin-2-yl]oxy}methyl)-3,5-dimethoxyphenoxy]phosphonic acid (see Scheme 1A)

A 2-hydroxyquinoxaline (HQ) hapten was modified with a caging group thatcould be released by alkaline phosphatase (AP), namely a caging groupcomprising a phosphatase enzyme substrate portion. Secondaryanti-species antibodies (goat-anti-rabbit and goat-anti-mouse) wereindividually labeled with either the caged hapten (cHQ) or AP. Thus, anypair of rabbit and mouse primary antibodies could be tested to determinetheir proximity to each other. The caged HQ was synthesized according tothe methodology set forth below and as illustrated in Scheme 1A, whichillustrates the preparation of caged HQ with a maleimide (MAL) reactivegroup for conjugation to antibodies (cHQ-MAL).

Compound 2. A soln. of 2,6-dimethoxy-4-hydroxybenzaldehyde (5.00 g, 27.4mmol) and triethylamine (4.17 g, 5.74 mL, 41.2 mmol) in EtOAc (25 mL)was cooled to 0° C. in an ice bath in stirring. Diethyl chlorophosphate(4.73 g, 27.4 mmol) was then added dropwise over a period of 5 min. Thereaction was allowed to warm to rt, followed by stirring at roomtemperature (“rt”) for about 6 h (check HPLC to confirm reaction wasgreater than about 90% complete). The reaction mixture was quenched byaddition of 1M HCl (100 mL) and the organic layer was separated andcollected. An additional quantity of EtOAc (100 mL) was added and theorganics were extracted with 1M HCl (2×100 mL), sat'd NaHCO₃ (3×100 mL)and brine (100 mL). The organic layer was dried over MgSO₄ and thesolvents removed under reduced pressure to give compound 2 as a lightbrown viscous oil (7.95 g, 91% yield).

Compound 3. A soln. of compound 2 (7.95 g, 25.0 mmol) in THE (about 100mL) was cooled to about 0° C. in an ice bath with stirring. NaBH₄ (1.42g, 37.5 mmol) was crushed to fine powder with a mortar and pestle andwas added portion-wise, followed by stirring at rt for about 4 h (checkHPLC to confirm reaction completion). The reaction mixture was carefullyquenched by addition of 1M HCl until bubbling ceased. The majority ofthe THF was then removed under reduced pressure. The resulting reactionmixture was extracted with EtOAc (3×100 mL). The organic layers werecollected and combined, followed by washing with brine (about 100 mL)and drying over MgSO₄. The solvents were then removed under reducedpressure. The resulting light brown viscous oil was dissolved in dryCH₂Cl₂ (about 50 mL) followed by cooling to about 0° C. in an ice bathunder an atmosphere of N₂ with stirring. SOCl₂ (4.46 g, 2.72 mL, 37.5mmol) was then added dropwise over a period of about 5 min. The reactionmixture was allowed to warm to rt, followed by stirring at rt for anadditional 1 h (check HPLC to confirm reaction completion). The reactionmixture was carefully quenched by addition of sat'd NaHCO₃ untilbubbling ceased. The resulting reaction mixture was extracted withCH₂Cl₂ (3×100 mL). The organic layers were collected and combined,followed by washing with brine (100 mL) and drying over MgSO₄. Thesolvents were then removed under reduced pressure to give compound 3 asan off-white low melting solid (7.54 g, 89% yield).

Compound 5. 3-Hydroxy-2-quinoxalinecarboxylic acid (3.00 g, 15.8 mmol)was dissolved in DMF (50 mL), followed by addition of4-dimethylaminopyridine (2.12 g, 17.4 mmol). The reaction mixture wasstirred at rt until the 4-dimethylaminopyridine dissolved, at whichpoint N,N′-disuccinimidyl carbonate (4.45 g, 17.4 mmol) was added. Thereaction mixture was stirred at rt for 30 min (Check HPLC to confirmreaction completion. If the reaction was not complete, additional 0.1 eqportions of DSC were added until HPLC showed complete conversion). Asoln. of N-Boc-ethylenediamine (3.04 g, 19.0 mmol) and triethylamine(2.40 g, 3.30 mL, 23.7 mmol) in DMF (10 mL) was then added, and thereaction was stirred at rt for 1 h (check HPLC for reaction completion).The reaction mixture was then poured onto a vigorously stirring soln. of1M HCl (250 mL). The resulting precipitate was collected by vacuumfiltration, washed several times with water, and dried underhigh-vacuum, giving compound 5 as a yellow solid (5.10 g, 97% yield).

Compound 6. Compound 5 (2.50 g, 7.52 mmol) was dissolved in DMF (50 mL),followed by addition of compound 3 (3.82 g, 11.3 mmol) and K₂CO₃ (5.20g, 37.6 mmol). The reaction vessel was sealed and heated to 55° C. in anoil bath with vigorous stirring for 2 h (Check HPLC to confirm reactioncompletion. If the reaction was not complete, additional 0.1 eq portionsof compound 3 were added until HPLC showed complete consumption ofcompound 5). The reaction mixture was filtered and the filtratecarefully quenched by addition of 1M HCl until bubbling ceased. Theresulting reaction mixture was extracted with EtOAc (3×100 mL). Theorganic layers were collected and combined, followed by washing withbrine (100 mL) and drying over MgSO₄. The solvents were then removedunder reduced pressure to give a light yellow viscous oil. The resultingresidue was purified by flash chromatography (Biotage Snap 50; hex:EA1:0 to 1:4) to give compound 6 as a light yellow solid (3.15 g, 66%yield).

Compound 7. Compound 6 (3.15 g, 4.96 mmol) was dissolved in dry CH₂C12(25 mL) followed by dropwise addition of trimethylsilyl bromide (3.80 g,3.28 mL, 24.8 mmol) over a period of 5 min. The vessel was sealed andthe reaction mixture was stirred at rt for 16 h (check HPLC to confirmreaction completion). MeOH (25 mL) was then added and the solvents wereremoved under reduced pressure. The resulting solid was triturated withCH₂Cl₂ (100 mL) and the resulting solid was collected by vacuumfiltration, giving compound 7 as a light yellow solid (2.06, 87% yield).

Compound 8. Compound 7 (2.06 g, 4.31 mmol) was suspended in DMF (10 mL),followed by addition of triethylamine (654 mg, 900 μL, 6.46 mmol) andfinally 3-maleimidopropionic acid NHS ester (1.15 g, 4.31 mmol). Thereaction vessel was sealed and the reaction mixture was vigorouslystirred at room temperature for about 4 h (check HPLC to confirmreaction completion). The reaction mixture was then diluted with MeOH(10 mL) and directly purified by prep RP-HPLC (0.05% TFA in H₂O:MeCN99:1 to 5:95 over 40 min) in 5 portions to give compound 8 as a lightyellow solid (1.95 g, 72% yield).

The maleimide functionalized HQ (cHQ-MAL) was conjugated to bothgoat-anti-rabbit and goat-anti-mouse IgG antibodies by twomethodologies, via disulfide or lysine groups. In the disulfide method,the IgG was reduced by dithiothreitol (DTT) and then treated with anexcess of cHQ-MAL. For the lysine method, the IgG was treated with anexcess of Traut's reagent (2-iminothiolane hydrochloride) before addingan excess of cHQ-MAL. In both cases the conjugates were purified by sizeexclusion chromatography (SEC) and characterized by gel electrophoresisand UV-Vis spectroscopy.

Example 2-Confirmation of Blocking of Caging Group

To confirm that the caging group of the caged hapten-antibody conjugatefrom Example 1 blocked an anti-hapten antibody from binding to therespective native (i.e. unmasked) hapten, the cHQ conjugate was testedwith single IHC detection. FIG. 5 illustrates an IHC staining schemewhere a single antigen (114) was detected with a secondary antibodyconjugated to cHQ, i.e. a cHQ caged hapten-antibody conjugate (103A).Following introduction of the cHQ caged hapten-antibody conjugate(103A), an AP enzyme (i.e. free, unconjugated AP) (113) was introducedto unmask the hapten of the caged hapten-antibody conjugate. Theresulting unmasked hapten (103B) was able to be recognized by ananti-hapten antibody (115). In some embodiments, the anti-haptenantibody (115) is conjugated to one or more enzymes (FIG. 5 illustratesthe conjugation to 3 HRP enzymes).

With reference to FIGS. 6A-6C, mouse-anti-PSA bound to its targetepitope on prostate tissue and was subsequently bound by goat-anti-mouseconjugated to cHQ (GAM-cHQ). Control slides (e.g. FIG. 6C) weregenerated using the goat-anti-mouse conjugated to uncaged HQ (GAM-HQ).Serial sections were exposed to two different conditions, namely (i)solutions with free (unconjugated) AP, pH adjust buffer and AP cofactors(magnesium salts); and (ii) solutions without AP, but with pH adjustbuffer and AP cofactors (magnesium salts). FIG. 6A illustrates tissuetreated with solution comprising AP; while FIG. 6B illustrates untreatedtissue, i.e. solution without AP. FIG. 6C illustrates a control slidewith a secondary antibody labeled with uncaged HQ. The sample tissuesthat were treated with AP under conditions favorable for the enzyme hadthe opportunity to allow unmasking of the caged hapten to occur (FIG.6A). The negative sample tissues (no AP) did not have the opportunityfor controlled unmasking to occur (FIG. 6B). These figures clearlyillustrate that the slide that was treated with a GAM-cHQ but was notexposed to AP shows no positive staining (FIG. 6B). However, the slidethat had both the GAM-cHQ and AP shows positive staining (FIG. 6A) andmatches the non-caged control slide (FIG. 6C). This tested both theeffectiveness of the caging group and its stability to the assayconditions.

Example 3-Measurement of Protein Proximity

A number of known positive and putative negative systems wereinvestigated to test the ability of the disclosed conjugates to enablemeasurement of protein proximity. E-cadherin and beta-catenin werechosen as known positive systems since the biology dictates that theseproteins are in close contact to each other in a normal cell.Her2/beta-catenin and EGFR/beta-catenin were chosen as pairs of proteinthat are in the same cell compartment (co-localized) but not expected tointeract with each other (not proximal) and therefore should notgenerate signal in any proximity assay. The antibody pairs were alsochosen so that there was one rabbit and one mouse primary antibody toallow the use of anti-species secondary detections.

With reference to the schemes outlined in FIGS. 2, 4, and 12, followingintroduction of the primary antibodies (e.g. an antibody conjugated to ahapten, a caged hapten-antibody conjugate, and an unmaskingenzyme-antibody conjugate) and secondary antibodies (e.g. an anti-haptenantibody conjugated to an enzyme), and subsequent unmasking of the cagedhapten, the uncaged HQ was detected with a mouse-anti-HQ conjugated tohorseradish peroxidase (HRP). At this point either DAB(3,3′-diaminobenzidine), silver or a tyramide-chromogen could be used tovisualize the proximity signal. A HRP/tyramide-hapten amplification stepcan also be included to boost the intensity (sensitivity) of the system(e.g. tyramide-HQ based optiView Amplification kit).

A general procedure for the automated caged hapten proximity assay wasused to test FFPE cases: The slides were deparaffinized using adeparaffinizing solution of DISCOVERY EZ Prep (Ventana, p/n 950-100) orDISCOVERY Wash (Ventana, p/n 950-510). Heat induced epitope retrievalwas performed on the slide using DISCOVERY CC1 (Ventana, p/n 950-124) at95° C. for a time dependent on the identity of the two primaryantibodies (0-92 minutes). DISCOVERY Inhibitor (Ventana, p/n 760-4840)was then applied to the slide to quench any endogenous peroxidase in thesample. A rabbit anti-target 1 antibody was co-incubated with a mouseanti-target 2 antibody at 37° C. for 16-32 minutes (dependent on theidentities of the primary antibodies). Then a 30 μg/mL goat anti-mousealkaline phosphatase conjugate was applied to the slide and incubatedfor 12 minutes. The slide was rinsed and a 20 μg/mL solution of thecaged hapten goat anti-rabbit conjugate was applied to the slide for 16minutes. After the incubation, a 500 mM tris buffer solution pH 10.0 anda 490 mM MgCl₂ solution were applied to the slide to enable thede-caging of the caged hapten. After the de-caging of the HQ, amouse-anti-HQ HRP (Ventana, p/n 760-4820) conjugate was applied to theslide to bind the uncaged HQ. In some cases the Amp HQ tyramideamplification kit (Ventana, p/n 760-052) was used to help boost thesignal intensity. Proximal proteins were visualized using the ChromomapDAB kit (Ventana, p/n 760-159), followed by counterstaining withHematoxylin II (Ventana, p/n 790-2208) and Bluing Reagent (Ventana, p/n760-2037).

FIGS. 7A-7C illustrate the results of this experiment and illustrateexamples of proteins with positive proximity. FIG. 7A illustrates asingle IHC DAB stain for Beta-Catenin (e.g. utilizing detectiontechniques comprising an antibody specific for Beta-Catenin, ananti-species antibody conjugated to an enzyme, and DAB). FIG. 7Billustrates a caged hapten proximity signal for Beta-Catenin andE-Cadherin (utilizing a caged hapten-antibody conjugate acted upon by anunmasking enzyme-antibody conjugate). FIG. 7C illustrates a single IHCDAB stain for E-Cadherin (e.g. utilizing detection techniques comprisingan antibody specific for E-Cadherin, an anti-species antibody conjugatedto an enzyme, and DAB).

FIGS. 8A-8C illustrates the results of IHC DAB staining and illustrateexamples of co-localized proteins without proximity as detected with theconjugates and methods described herein. FIG. 8A illustrates a singleIHC DAB stain for EGFR; FIG. 8B illustrates the absence of a cagedhapten proximity signal for EGFR and E-Cadherin; and FIG. 8C illustratesa single IHC DAB stain for E-Cadherin.

In addition, FIGS. 9A and 9B illustrate examples of co-localizedproteins with and without proximity on a different tissue type. Here,FIG. 9A illustrates positive caged hapten proximity signal forBeta-catenin and E-Cadherin, while FIG. 9B illustrates the lack of acaged hapten proximity signal for Beta-catenin and Her2. These examplesillustrate that the assay is applicable to different tissue types, butgenerates the same type of results.

The signal intensity output from the above-described assays may be“dialed-in” a number of ways. The use of on-market signal amplificationis easily applied to the presently disclosed technology and is believedto easily boost the signal (see, e.g. the amplification techniquesdescribed in U.S. Publication No. 2012/0171668, and PCT/EP2015/0533556,the disclosures of which are hereby incorporated by reference herein).

As illustrated in FIGS. 10A and 10B, the number of caged haptensconjugated to the secondary antibody also allows the sensitivity of theassay to be adjusted to fit a given system. In this example, FIG. 10Aillustrates a sample labeled with goat-anti-rabbit and 4 (four) cHQlabels while FIG. 10B illustrates a sample labeled with goat-anti-rabbitand 9 (nine) cHQ labels. Here, the comparative increase in signalintensity is based on the increased number of caged haptens conjugatedto the secondary antibody, where FIG. 10B clearly shows the increase instaining intensity as compared with FIG. 10A.

Example 4-Detection with Different HRP Systems

Different HRP systems providing different detectable signals (e.g.colors) were tested. For example, FIGS. 11A and 11B illustrate detectionwith silver (11A) and Tyramide-TAMRA (11B). Both systems were able todetect proximal protein targets in a sample.

Example 5-Multiplex Detection of Total Protein and Proximal ProteinSignal

FIG. 12 is a schematic illustrating the multiplex detection of one totalprotein (Target 2, AP-Yellow) and the proximal protein signal (Target1+Target 2, HRP-Silver). Any type of HRP detection system may be used toshow the proximity signal and any AP detection system (e.g. naphtholphosphate/Fast Red, BCIP/NBT, quinone methide (QM)-chromogen) may beused to visualize the total protein.

The detection of proximal proteins utilizing HRP-DAB (FIG. 13A) andHRP-Silver with the addition of a total protein stain for one of thetargets has also been demonstrated (FIG. 13B). These experiments wereperformed with E-cadherin and Beta-catenin on tonsil tissue. Inaddition, FIGS. 14A and 14B illustrate the use of different HRPdetection systems to visualize the proximity signal along with totalprotein, utilizing an E-cadherin and Beta-catenin duel stain. FIG. 14Aillustrates proximity detected with HRP-Purple and Beta-Catenin detectedwith AP-Yellow, while FIG. 13B illustrates proximity detected withHRP-Silver and Beta-Catenin detected with AP-Yellow.

Example 6-PD1/PD-L1

Binding of the ligand PD-L1 to its receptor PD-1 modulates theactivation or inhibition of T-cells. Tumor cells can use thisinteraction to evade the immune system. Detection of a PD-L1/PD-1complex may provide a better indication of the blockade status of theimmune checkpoint within a tumor than simple measurement of eitherprotein by itself.

FIG. 16 illustrates a caged hapten proximity signal for PD-1 and PD-L1(utilizing a caged hapten-antibody conjugate acted upon by an unmaskingenzyme-antibody conjugate) in a NSCLC. The purple signal represents thedetection of PD-L1/PD-1 complexes.

The caged hapten proximity assay was used to test FFPE cases of NSCLCand tonsil. The cases were run on the DISCOVERY ULTRA instrument, fullyautomated. The slides were deparaffinized using a deparaffinizingsolution of DISCOVERY EZ Prep (Ventana, p/n 950-100) or DISCOVERY Wash(Ventana, p/n 950-510). Heat induced epitope retrieval was performed onthe slide using DISCOVERY CC1 (Ventana, p/n 950-124) at 95° C. for 64minutes. DISCOVERY Inhibitor (Ventana, p/n 760-4840) was then applied tothe slide to quench any endogenous peroxidase in the sample. Rabbitanti-PD-L1 (SP263) (Ventana, p/n 790-4905) was co-incubated with a mouseanti-PD-1 (NAT105) (Ventana, p/n 760-4895) for 32 minutes at 37° C. Fora negative control the mouse anti-PD-1 was omitted. For the remainder ofthe run, the slide was incubated at 37° C. A 30 μg/mL goat anti-mousealkaline phosphatase conjugate was applied to the slide and incubatedfor 12 minutes. The slide was rinsed and a 20 μg/mL solution of thecaged hapten goat anti-rabbit conjugate was applied to the slide for 16minutes. After the incubation, a 500 mM tris buffer solution pH 10.0 anda 490 mM MgCl₂ solution were applied to the slide to enable thede-caging of the caged hapten. After the de-caging of the HQ, amouse-anti-HQ HRP (Ventana, p/n 760-4820) conjugate was applied to theslide to bind the uncaged HQ. Proximal proteins were visualized usingthe DISCOVERY Purple kit (Ventana, p/n 760-229). The Amp HQ tyramideamplification kit (Ventana, p/n 760-052) was used to help boost thesignal intensity when necessary.

FIG. 17 illustrates a caged hapten proximity signal for PD-L1 and PD-1along with the total protein detection for PD-1. After detection ofproximal proteins with DISCOVERY Purple then the uncaging enzyme(alkaline phosphatase) was detected with DISCOVERY Yellow (Ventana, p/n760-239) to visualize the PD-1 on the tissue.

FIG. 18 illustrates a negative control for a caged hapten proximitysignal for PD-L1 and PD-1 in tonsil tissue. The primary antibody forPD-1 was omitted from the assay. No signal was observed demonstratingthe specificity of the detection reagents for the protein complex.

In all cases the slides were counterstained with Hematoxylin II(Ventana, p/n 790-2208) and Bluing Reagent (Ventana, p/n 760-2037).

Example 7-PD1/PD-L1 Combined with CD8

FIG. 19 illustrates an example of combining the caged hapten proximitysignal for PD-L1 and PD-1 with the subsequent detection of anotherbiomarker in tonsil tissue. In this case CD8 was detected after thePD-L1/PD1 complex to generate an assay that visualized the CD8 cellsthat were involved in PD-L1/PD-1 interactions. For multiplexing, thePD-1 and PD-L1 proximity was first detected as previously described. Theslides were heated to 90° C. for 8 minutes to assist in the elution ofthe previously bound primaries. Rabbit anti-CD8 (SP57) (Ventana, p/n790-4460) was applied to the slide and incubated for 16 minutes at 37°C. The anti-CD8 was detected using UltraMap Rb AP (Ventana, p/n760-4314) followed by the DISCOVERY Yellow chromogen. The slides werecounterstained with Hematoxylin II (Ventana, p/n 790-2208) and BluingReagent (Ventana, p/n 760-2037).

Example 8-Her2/Her3

The caged hapten proximity assay was used to test FFPE cases of BT-474cell lines. The cases were run on the DISCOVERY ULTRA instrument, fullyautomated. The slides were deparaffinized using a deparaffinizingsolution of DISCOVERY EZ Prep (Ventana, p/n 950-100) or DISCOVERY Wash(Ventana, p/n 950-510). Heat induced epitope retrieval was performed onthe slide using DISCOVERY CC1 (Ventana, p/n 950-124) at 95° C. for 64minutes. DISCOVERY Inhibitor (Ventana, p/n 760-4840) was then applied tothe slide to quench any endogenous peroxidase in the sample. Rabbitanti-Her2 (4B5) (Ventana, p/n 790-2991) was co-incubated with a mouseanti-Her3 (SPM738) (Spring, p/n E19260) for 32 minutes at 37° C. Then a30 μg/mL goat anti-mouse alkaline phosphatase conjugate was applied tothe slide and incubated for 12 minutes. The slide was rinsed and a 20μg/mL solution of the caged hapten goat anti-rabbit conjugate wasapplied to the slide for 16 minutes. After the incubation, a 500 mM trisbuffer solution pH 10.0 and a 490 mM MgCl₂ solution were applied to theslide to enable the de-caging of the caged hapten. After the de-cagingof the HQ, a mouse-anti-HQ HRP (Ventana, p/n 760-4820) conjugate wasapplied to the slide to bind the uncaged HQ. The Amp HQ tyramideamplification kit (Ventana, p/n 760-052) was used to help boost thesignal intensity. Proximal proteins were visualized using the ChromomapDAB kit (Ventana, p/n 760-159), followed by counterstaining withHematoxylin II (Ventana, p/n 790-2208) and Bluing Reagent (Ventana, p/n760-2037).

FIGS. 20A-20C illustrate the results of this experiment and illustrateexamples of proteins with positive proximity. FIG. 20A illustrates asingle IHC DAB stain for Her2 (e.g. utilizing detection techniquescomprising an antibody specific for Her2, an anti-species antibodyconjugated to an enzyme, and DAB). FIG. 20B illustrates a caged haptenproximity signal for Her2 and Her3 (utilizing a caged hapten-antibodyconjugate acted upon by an unmasking enzyme-antibody conjugate). FIG.20C illustrates a single IHC DAB stain for Her3 (e.g. utilizingdetection techniques comprising an antibody specific for Her3, ananti-species antibody conjugated to an enzyme, and DAB).

Example 9-Measurement of Protein Proximity

A number of known positive and putative negative systems wereinvestigated to test the ability of the disclosed conjugates to enablemeasurement of protein proximity. E-cadherin and beta-catenin werechosen as known positive systems since the biology dictates that theseproteins are in close contact to each other in a normal cell. Theantibody pair was also chosen so that there was one rabbit and one mouseprimary antibody to allow the use of anti-species secondary detections.

With reference to the schemes outlined in FIG. 21, followingintroduction of the primary antibodies and secondary antibodies (e.g. ananti-species antibody conjugated to an enzyme and an anti-speciesantibody conjugated to a caged hapten), and subsequent unmasking of thecaged hapten, the uncaged NP was detected with a mouse-anti-NPconjugated to horseradish peroxidase (HRP). At this point either DAB(3,3′-diaminobenzidine), silver or a tyramide-chromogen could be used tovisualize the proximity signal. A HRP/tyramide-hapten amplification stepcan also be included to boost the intensity (sensitivity) of the system(e.g. tyramide-HQ based optiView Amplification kit).

A general procedure for the automated caged hapten proximity assay wasused to test FFPE cases: The slides were deparaffinized using adeparaffinizing solution of DISCOVERY EZ Prep (Ventana, p/n 950-100) orDISCOVERY Wash (Ventana, p/n 950-510). Heat induced epitope retrievalwas performed on the slide using DISCOVERY CC1 (Ventana, p/n 950-124) at95° C. for a time dependent on the identity of the two primaryantibodies (0-92 minutes). DISCOVERY Inhibitor (Ventana, p/n 760-4840)was then applied to the slide to quench any endogenous peroxidase in thesample. A rabbit anti-target 1 antibody was co-incubated with a mouseanti-target 2 antibody at 37° C. for 16-32 minutes (dependent on theidentities of the primary antibodies). Then a 30 μg/mL goat anti-mousealkaline phosphatase conjugate was applied to the slide and incubatedfor 12 minutes. The slide was rinsed and a 20 μg/mL solution of thecaged hapten goat anti-rabbit conjugate was applied to the slide for 16minutes. After the incubation, a 500 mM tris buffer solution pH 10.0 anda 490 mM MgCl₂ solution were applied to the slide to enable thede-caging of the caged hapten. After the de-caging of the NP, amouse-anti-NP HRP (Ventana, p/n 760-4820) conjugate was applied to theslide to bind the uncaged HQ. In some cases the Amp HQ tyramideamplification kit (Ventana, p/n 760-052) was used to help boost thesignal intensity. Proximal proteins were visualized using the ChromomapDAB kit (Ventana, p/n 760-159), followed by counterstaining withHematoxylin II (Ventana, p/n 790-2208) and Bluing Reagent (Ventana, p/n760-2037).

The caged hapten proximity assay was used to test FFPE cases of tonsil.The cases were run on the VENTANA DISCOVERY ULTRA or VENTANA BenchmarkXT instrument, fully automated. The slides were deparaffinized using adeparaffinizing solution of DISCOVERY EZ Prep (Ventana, p/n 950-100) orDISCOVERY Wash (Ventana, p/n 950-510). Heat induced epitope retrievalwas performed on the slide using DISCOVERY CC1 (Ventana, p/n 950-124) at95° C. for 64 minutes. DISCOVERY Inhibitor (Ventana, p/n 760-4840) wasthen applied to the slide to quench any endogenous peroxidase in thesample. Rabbit anti-E-Cadherin (EP700Y) (Ventana, p/n 760-4440) wasco-incubated with a mouse anti-β-Catenin (14) (Ventana, p/n 760-4242)for 32 minutes at 37° C. A 30 μg/mL goat anti-mouse alkaline phosphataseconjugate was applied to the slide and incubated for 12 minutes. Theslide was rinsed and a 20 μg/mL solution of the caged hapten goatanti-rabbit conjugate was applied to the slide for 16 minutes. After theincubation, a 500 mM tris buffer solution pH 10.0 and a 490 mM MgCl₂solution were applied to the slide to enable the de-caging of the cagedhapten. After the de-caging of the NP, a mouse-anti-NP HRP conjugate wasapplied to the slide to bind the uncaged NP. The Amp HQ tyramideamplification kit (Ventana, p/n 760-052) was used to boost the signalintensity. Proximal proteins were visualized using the Chromomap DAB kit(Ventana, p/n 760-159), followed by counterstaining with Hematoxylin II(Ventana, p/n 790-2208) and Bluing Reagent (Ventana, p/n 760-2037).

FIGS. 22A-22B illustrate the results of this experiment and illustrateexamples of proteins with positive proximity. Both figures illustrate acaged hapten proximity signal for Beta-Catenin and E-Cadherin (utilizinga caged hapten-antibody conjugate acted upon by an unmaskingenzyme-antibody conjugate). FIG. 22A illustrates the positive proximitysignal observed with a caged hapten of Formula (IC) or (ID), while FIG.22B illustrates the positive proximity signal observed with a cagedhapten of Formula (IE) or (IF). No difference in specific signal isobserved between the two samples.

Example 10-Confirmation of Blocking of Caging Group

To confirm that the caging group of the caged hapten-antibody conjugateis present and blocking an anti-hapten antibody from binding to therespective native (i.e. uncaged) hapten, the cNP conjugate was testedwith single IHC detection. To detect very low levels of uncaging events,biomarkers with very high expression levels were selected on FFPEsamples with very high expression levels (e.g. Lambda protein on tonsiltissue and Her2 protein on SKBR3 cell lines). FIG. 3 illustrates an IHCstaining scheme where a single antigen (101) was detected with asecondary antibody conjugated to cNP, i.e. a cNP caged hapten-antibodyconjugate (105). Following introduction of the cNP caged hapten-antibodyconjugate (105), an anti-hapten antibody (109) was introduced. In someembodiments, the anti-hapten antibody (109) is conjugated to one or moreenzymes (FIG. 3 illustrates the conjugation to 3 HRP enzymes).

The cases were run on the VENTANA DISCOVERY ULTRA or VENTANA BenchmarkXT instrument, fully automated. The slides were deparaffinized using adeparaffinizing solution of DISCOVERY EZ Prep (Ventana, p/n 950-100) orDISCOVERY Wash (Ventana, p/n 950-510). For the lambda assay enzymaticepitope retrieval was performed on the slide using Protease 1 (Ventana,p/n 760-2018) at 37° C. for 4 minutes. For the Her2 assay heat inducedepitope retrieval was performed on the slide using DISCOVERY CC1(Ventana, p/n 950-124) at 95° C. for 64 minutes. DISCOVERY Inhibitor(Ventana, p/n 760-4840) was then applied to the slide to quench anyendogenous peroxidase in the sample. Rabbit anti-Lambda (Ventana, p/n760-2515) or a rabbit-anti-Her2 (Ventana, p/n 760-2991) was incubatedfor 32 minutes at 37° C. The slide was rinsed and a 20 μg/mL solution ofthe caged hapten goat anti-rabbit conjugate was applied to the slide for16 minutes. After the incubation, a mouse-anti-NP HRP conjugate wasapplied to the slide to bind any uncaged NP. The Amp HQ tyramideamplification kit (Ventana, p/n 760-052) was used to boost the signalintensity. Proximal proteins were visualized using the Chromomap DAB kit(Ventana, p/n 760-159), followed by counterstaining with Hematoxylin II(Ventana, p/n 790-2208) and Bluing Reagent (Ventana, p/n 760-2037).

With reference to FIGS. 24A and 24C, rabbit-anti-Her2(4B5) bound to itstarget epitope on SKBR3 cells and was subsequently bound bygoat-anti-rabbit conjugated to a caged happen of Formula (IC)-(IF) cNP(GAR-cNP) (see, for example, the conjugates of Formula (IVA)-(IVD). FIG.4A illustrates tissue treated with a caged hapten of Formula (IC) or(ID) and the observed signal indicates incomplete caging. FIG. 4Cillustrates tissue treated with a caged happen of Formula (IE) or (IF)and the absence of any signal indicates complete caging.

With reference to FIGS. 24B and 24D, rabbit-anti-Lambda bound to itstarget epitope on tonsil tissue and was subsequently bound bygoat-anti-rabbit conjugated to a caged happen of Formula (IC)-(IF) cNP(GAR-cNP). FIG. 4B illustrates tissue treated with a caged hapten ofFormula (IC) or (ID) and the observed signal indicates incompletecaging. FIG. 4D illustrates tissue treated with a caged hapten ofFormula (IE) or (IF) and the absence of any signal indicates completecaging.

Example 11-Stability of the Caging Group

Applicants have discovered that certain caged haptens of Formulas (IE)and (IF) have enhanced stability as compared with caged haptens ofFormulas (IC) or (ID). For example, and with reference to FIG. 25, itwas unexpectedly found that compounds 8, 13a, and 13b had enhancedstability as compared with compound 14. As between compounds 8 and 14,and without wishing to be bound by any particular theory, it is believedthat the addition of a —O-alkyl group between the leaving group and theenzyme substance increases the stability of the caged hapten. Indeed, asmeasured at about day 65, for compound 14 the percent decaging was about20%, whereas for compound 8 the percent decaging was less than 10%. Forcompounds 13a and 13b, which do not include an aryl leaving group, thepercent decaging after about 65 days was about 4.5% and about 2.5%,respectively. While these amounts of decaging might not be orders ofmagnitude in difference, even smaller differences are important in thediagnostics industry, where false positive results could severely hinderthe subsequent patient care provided by a medical practioner.

Indeed, Applicants submit that a reduction in the amount of decagingwill decrease the amount of false positive results. By way of example,when the caged hapten of compound 14 is stored for about 65 days, acertain percentage of the caged hapten will “lose” its caging group. Theskilled artisan will appreciate that caged haptens lacking a caginggroup will be in solution with intact caged hapten molecules andintroduced to a biological sample contemporaneously. The antibodyportions of caged haptens lacking the caging group will bind targets,thus labeling the targets with haptens (which in turn may be recognizedwith anti-hapten antibodies) (see, for example, FIG. 23). The skilledartisan will appreciate that the labeling of targets with such cagedhaptens lacking caging groups may result in false positives in anyprotein proximity assay (see the mechanism of action of de-caging acaged hapten as illustrated in FIG. 2). By reducing the amount of cagedhapten lacking a caging group, it is possible to reduce, mitigate, orprevent the generation of false positive signals.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet are incorporated herein by reference, intheir entirety. Aspects of the embodiments can be modified, if necessaryto employ concepts of the various patents, applications and publicationsto provide yet further embodiments.

Although the present disclosure has been described with reference to anumber of illustrative embodiments, it should be understood thatnumerous other modifications and embodiments can be devised by thoseskilled in the art that will fall within the spirit and scope of theprinciples of this disclosure. More particularly, reasonable variationsand modifications are possible in the component parts and/orarrangements of the subject combination arrangement within the scope ofthe foregoing disclosure, the drawings, and the appended claims withoutdeparting from the spirit of the disclosure. In addition to variationsand modifications in the component parts and/or arrangements,alternative uses will also be apparent to those skilled in the art.

Additional Embodiments

-   Additional Embodiment 1. A method for analyzing a sample to    determine whether a first target is proximal to a second target, the    method comprising:    -   (a) contacting the sample with a second unmasking        enzyme-antibody conjugate to form a second target-unmasking        enzyme-antibody conjugate complex;    -   (b) contacting the sample with a first caged hapten-antibody        conjugate to form a first target-caged hapten-antibody conjugate        complex;    -   (c) unmasking the caged hapten of the first target-caged        hapten-antibody conjugate complex to form a first        target-unmasked hapten-antibody conjugate complex;    -   (d) contacting the sample with first detection reagents to label        the first target-unmasked hapten-antibody conjugate complex or        the first target; and    -   (e) detecting the labeled first target-unmasked hapten-antibody        conjugate complex or labeled first target,

wherein the first caged hapten-antibody conjugate has the structure ofFormulas (IVC) or (IVD):

-   -   wherein    -   “Antibody” is an antibody;    -   “Hapten” is a hapten;    -   A is a group comprising a branched or unbranched, substituted or        unsubstituted, saturated or unsaturated aliphatic group having        between 1 and 15 carbon atoms, and optionally having one or more        heteroatoms selected from the group consisting of O, N, or S;    -   W is a 5-, 6-, or 7-membered substituted or unsubstituted        aromatic or heterocyclic group;    -   Z is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S,    -   each R¹ is independently selected from H, F, Cl, Br, I,        —O-methyl, —O— ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl,        —O-sec-butyl, or —O-iso-butyl; a —S-alkyl group having between 1        and 4 carbon atoms; an —O-alkyl group having between 1 and 4        carbon atoms; a —N(H)-alkyl group having between 1 and 4 carbon        atoms; a —N-(alkyl)₂ group having between 1 and 6 carbon atoms;        an alkyl group having between 1 and 4 carbon atoms and        optionally substituted with N or S; cyano groups; and carboxyl        groups;    -   V is a bond, a substituted or unsubstituted alkyl group having        between 1 and 4 carbon atoms, or a substituted or unsubstituted        —O-alkyl group;    -   R³ is an enzyme substrate;    -   n is an integer ranging from 1 to 25, and    -   q is 0 or 1;    -   s is an integer ranging from 1 to 4; and    -   t is 0 or 1.

-   Additional Embodiment 2. The method of additional embodiment 1,    wherein the first detection reagents comprise (i) a secondary    antibody specific to the unmasked hapten of the first    target-unmasked hapten-antibody complex, the secondary antibody    conjugated to a first enzyme such that the secondary antibody labels    the first target-unmasked hapten-antibody complex with the first    enzyme; and (ii) a first substrate for the first enzyme.

-   Additional Embodiment 3. The method of additional embodiment 2,    wherein the first substrate is a chromogenic substrate or a    fluorescent substrate.

-   Additional Embodiment 4. The method of additional embodiment 1,    wherein the first detection reagents include amplification    components to label the unmasked enzyme of the first target-unmasked    hapten-antibody conjugate complex with a plurality of first reporter    moieties.

-   Additional Embodiment 5. The method of additional embodiment 4,    wherein the plurality of first reporter moieties are haptens.

-   Additional Embodiment 6. The method of additional embodiment 5,    wherein the first detection reagents further comprise secondary    antibodies specific to the plurality of first reporter moieties,    each secondary antibody conjugated to a second reporter moiety.

-   Additional Embodiment 7. The method of additional embodiment 5,    wherein the second reporter moiety is selected from the group    consisting of an amplification enzyme or a fluorophore.

-   Additional Embodiment 8. The method of additional embodiment 7,    wherein the second reporter moiety is an amplification enzyme and    wherein the first detection reagents further comprise a first    chromogenic substrate or a fluorescent substrate for the    amplification enzyme.

-   Additional Embodiment 9. The method of additional embodiment 1,    further comprising contacting the sample with a second substrate    specific for the unmasking enzyme of the second target-unmasking    enzyme-antibody conjugate complex and detecting signals    corresponding to a product of a reaction between the second    substrate and the unmasking enzyme.

-   Additional Embodiment 10. A method for analyzing a sample to    determine whether a first target is proximal to a second target, the    method comprising:    -   (a) contacting the sample with a second unmasking        enzyme-antibody conjugate to form a second target-unmasking        enzyme-antibody conjugate complex;    -   (b) contacting the sample with a first caged hapten-antibody        conjugate to form a first target-caged hapten-antibody conjugate        complex;    -   (c) unmasking the caged hapten of the first target-caged        hapten-antibody conjugate complex to form a first        target-unmasked hapten-antibody conjugate complex;    -   (d) performing a signal amplification step to label the first        target-unmasked hapten-antibody conjugate complex with a        plurality of reporter moieties; and    -   (e) detecting the plurality of reporter moieties, wherein the        first caged hapten-antibody conjugate has the structure of        Formulas (IVC) or (IVD):

-   -   wherein    -   “Antibody” is an antibody;    -   “Hapten” is a hapten;    -   A is a group comprising a branched or unbranched, substituted or        unsubstituted, saturated or unsaturated aliphatic group having        between 1 and 15 carbon atoms, and optionally having one or more        heteroatoms selected from the group consisting of O, N, or S;    -   W is a 5-, 6-, or 7-membered substituted or unsubstituted        aromatic or heterocyclic group;    -   Z is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S,    -   each R¹ is independently selected from H, F, Cl, Br, I,        —O-methyl, —O— ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl,        —O-sec-butyl, or —O-iso-butyl; a —S-alkyl group having between 1        and 4 carbon atoms; an —O-alkyl group having between 1 and 4        carbon atoms; a —N(H)-alkyl group having between 1 and 4 carbon        atoms; a —N-(alkyl)₂ group having between 1 and 6 carbon atoms;        an alkyl group having between 1 and 4 carbon atoms and        optionally substituted with N or S; cyano groups; and carboxyl        groups;    -   V is a bond, a substituted or unsubstituted alkyl group having        between 1 and 4 carbon atoms, or a substituted or unsubstituted        —O-alkyl group;    -   R³ is an enzyme substrate;    -   n is an integer ranging from 1 to 25, and    -   q is 0 or 1;    -   s is an integer ranging from 1 to 4; and    -   t is 0 or 1.

-   Additional Embodiment 11. The method of additional embodiment 10,    wherein the plurality of reporter moieties are haptens; and wherein    the method further comprises introducing secondary antibodies    specific to the plurality of first reporter moieties, each secondary    antibody conjugated to a second reporter moiety.

-   Additional Embodiment 12. The method of additional embodiment 11,    wherein the second reporter moiety is an amplification enzyme and    wherein the method further comprises introducing a chromogenic    substrate or a fluorescent substrate for the amplification enzyme.

-   Additional Embodiment 13. The method of additional embodiment 10,    further comprising detecting a total amount of target in the sample.

-   Additional Embodiment 14. A method for analyzing a sample to    determine whether a first target is proximal to a second target, the    method comprising:    -   (a) contacting the sample with a second unmasking        enzyme-antibody conjugate to form a second target-unmasking        enzyme-antibody conjugate complex;    -   (b) contacting the sample with a first caged hapten-antibody        conjugate to form a first target-caged hapten-antibody conjugate        complex;    -   (c) performing a decaging step such that an unmasking enzyme of        the second target-unmasking enzyme-antibody conjugate complex        reacts with an enzyme substrate portion of the first        target-caged hapten-antibody conjugate complex to form a first        target-unmasked hapten-antibody conjugate complex;    -   (d) contacting the sample with first detection reagents to label        the first target-unmasked hapten-antibody conjugate complex or        the first target; and    -   (e) detecting the labeled first target-unmasked hapten-antibody        conjugate complex or labeled first target,

wherein the first caged hapten-antibody conjugate has the structure ofFormulas (IVC) or (IVD):

-   -   wherein    -   “Antibody” is an antibody;    -   “Hapten” is a hapten;    -   A is a group comprising a branched or unbranched, substituted or        unsubstituted, saturated or unsaturated aliphatic group having        between 1 and 15 carbon atoms, and optionally having one or more        heteroatoms selected from the group consisting of O, N, or S;    -   W is a 5-, 6-, or 7-membered substituted or unsubstituted        aromatic or heterocyclic group;    -   Z is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S;    -   each R¹ is independently selected from H, F, Cl, Br, I,        —O-methyl, —O— ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl,        —O-sec-butyl, or —O-iso-butyl; a —S-alkyl group having between 1        and 4 carbon atoms; an —O-alkyl group having between 1 and 4        carbon atoms; a —N(H)-alkyl group having between 1 and 4 carbon        atoms; a —N-(alkyl)₂ group having between 1 and 6 carbon atoms;        an alkyl group having between 1 and 4 carbon atoms and        optionally substituted with N or S; cyano groups; and carboxyl        groups;    -   V is a bond, a substituted or unsubstituted alkyl group having        between 1 and 4 carbon atoms, or a substituted or unsubstituted        —O-alkyl group;    -   R³ is an enzyme substrate;    -   n is an integer ranging from 1 to 25, and    -   q is 0 or 1;    -   s is an integer ranging from 1 to 4; and    -   t is 0 or 1.

-   Additional Embodiment 15. The method of additional embodiment 14,    wherein the decaging step comprises changing the temperature of the    sample.

-   Additional Embodiment 16. The method of additional embodiment 14,    wherein the decaging step comprises altering a pH of the sample.

-   Additional Embodiment 17. The method of additional embodiment 14,    wherein the decaging step comprises introducing one or more washing    steps.

-   Additional Embodiment 18. The method of additional embodiment 14,    wherein the decaging step comprises adding cofactors for the    unmasking enzyme.

-   Additional Embodiment 19. The method of additional embodiment 14,    further comprising detecting a total amount of target in the sample.

-   Additional Embodiment 20. A method for analyzing a sample to    determine whether a first target is proximal to a second target, the    method comprising:    -   (a) contacting the sample with a caged hapten-antibody conjugate        specific to the first target to form a first target-caged        hapten-antibody conjugate complex;    -   (b) contacting the sample with an unmasking enzyme-antibody        conjugate specific to the second target to form a second        target-unmasking enzyme-antibody conjugate complex, wherein an        unmasking enzyme of the unmasking enzyme-antibody conjugate is        selected such that it is capable of reacting with an enzyme        substrate portion of the caged hapten-antibody conjugate to form        a first target-unmasked hapten-antibody conjugate complex;    -   (c) contacting the sample with first detection reagents to label        the first target-unmasked hapten-antibody conjugate complex or        the first target; and    -   (d) detecting the labeled first target-unmasked hapten-antibody        conjugate complex or labeled first target,

wherein the first caged hapten-antibody conjugate has the structure ofFormulas (IVC) or (IVD):

-   -   wherein    -   “Antibody” is an antibody;    -   “Hapten” is a hapten;    -   A is a group comprising a branched or unbranched, substituted or        unsubstituted, saturated or unsaturated aliphatic group having        between 1 and 15 carbon atoms, and optionally having one or more        heteroatoms selected from the group consisting of O, N, or S;    -   W is a 5-, 6-, or 7-membered substituted or unsubstituted        aromatic or heterocyclic group;    -   Z is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S;    -   each R¹ is independently selected from H, F, Cl, Br, I,        —O-methyl, —O— ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl,        —O-sec-butyl, or —O-iso-butyl; a —S-alkyl group having between 1        and 4 carbon atoms; an —O-alkyl group having between 1 and 4        carbon atoms; a —N(H)-alkyl group having between 1 and 4 carbon        atoms; a —N-(alkyl)₂ group having between 1 and 6 carbon atoms;        an alkyl group having between 1 and 4 carbon atoms and        optionally substituted with N or S; cyano groups; and carboxyl        groups;    -   V is a bond, a substituted or unsubstituted alkyl group having        between 1 and 4 carbon atoms, or a substituted or unsubstituted        —O-alkyl group;    -   R³ is an enzyme substrate;    -   n is an integer ranging from 1 to 25, and    -   q is 0 or 1;    -   s is an integer ranging from 1 to 4; and    -   t is 0 or 1.

-   Additional Embodiment 21. The method of additional embodiment 20,    wherein a caged hapten portion of the caged hapten-antibody    conjugate is derived from a hapten selected from the group    consisting of DCC, biotin, nitropyrazole, thiazolesulfonamide,    benzofurazan, and 2-hydroxyquinoxaline.

-   Additional Embodiment 22. The method of additional embodiment 20,    wherein the unmasking enzyme of the unmasking enzyme-antibody    conjugate is selected from the group consisting of alkaline    phosphatase, B-glucosidase, B-Galactosidase, B-Glucuronidase,    Lipase, Sulfatase, Amidase, Protease, Nitroreductase, beta-lactamase    & neuraminidase, and Urease.

-   Additional Embodiment 23. The method of additional embodiment 20,    wherein the first detection reagents comprise (i) a secondary    antibody specific to the unmasked hapten of the first    target-unmasked hapten-antibody complex, the secondary antibody    conjugated to a first enzyme such that the secondary antibody labels    first target-unmasked hapten-antibody complex with the first enzyme;    and (ii) a first chromogenic substrate or fluorescent substrate.

-   Additional Embodiment 24. The method of additional embodiment 23,    wherein the first enzyme is different than the unmasking enzyme.

-   Additional Embodiment 25. The method of additional embodiment 24,    wherein the first enzyme is a peroxidase.

-   Additional Embodiment 26. The method of additional embodiment 23,    wherein the first chromogenic substrate is selected from the group    consisting of 3,3′-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole    (AEC), HRP-Silver, and tyramide-chromogens.

-   Additional Embodiment 27. The method of additional embodiment 26,    further comprising contacting the sample with a second chromogenic    substrate or fluorescent substrate specific for the unmasking enzyme    of the second target-unmasking enzyme-antibody conjugate complex,    wherein the first and second chromogenic substrates are different.

-   Additional Embodiment 28. The method of additional embodiment 20,    wherein the first detection reagents include components to amplify    the amount of label introduced to the first target-unmasked    hapten-antibody conjugate complex.

-   Additional Embodiment 29. The method of additional embodiment 20,    wherein the first target is one of PD-1 or PD-L1, and the second    target is the other of PD-1 or PD-L1.

-   Additional Embodiment 30. A method for analyzing a sample to    determine whether a first target is proximal to a second target, the    method comprising:    -   (a) contacting the sample with a first detection probe, the        first detection probe comprising one of a caged hapten-antibody        conjugate or an unmasking enzyme-antibody conjugate;    -   (b) contacting the sample with a second detection probe, the        second detection probe comprising the other of the caged        hapten-antibody conjugate or the unmasking enzyme-antibody        conjugate;    -   (c) contacting the sample with at least first detection reagents        to label a formed unmasked hapten-antibody conjugate target        complex; and    -   (d) detecting signals from the labeled unmasked hapten-antibody        conjugate target complex,

wherein the first caged hapten-antibody conjugate has the structure ofFormulas (IVC) or (IVD):

-   -   wherein    -   “Antibody” is an antibody;    -   “Hapten” is a hapten;    -   A is a group comprising a branched or unbranched, substituted or        unsubstituted, saturated or unsaturated aliphatic group having        between 1 and 15 carbon atoms, and optionally having one or more        heteroatoms selected from the group consisting of O, N, or S;    -   W is a 5-, 6-, or 7-membered substituted or unsubstituted        aromatic or heterocyclic group;    -   Z is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S;    -   each R¹ is independently selected from H, F, Cl, Br, I,        —O-methyl, —O— ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl,        —O-sec-butyl, or —O-iso-butyl; a —S-alkyl group having between 1        and 4 carbon atoms; an —O-alkyl group having between 1 and 4        carbon atoms; a —N(H)-alkyl group having between 1 and 4 carbon        atoms; a —N-(alkyl)₂ group having between 1 and 6 carbon atoms;        an alkyl group having between 1 and 4 carbon atoms and        optionally substituted with N or S; cyano groups; and carboxyl        groups;    -   V is a bond, a substituted or unsubstituted alkyl group having        between 1 and 4 carbon atoms, or a substituted or unsubstituted        —O-alkyl group;    -   R³ is an enzyme substrate;    -   n is an integer ranging from 1 to 25, and    -   q is 0 or 1;    -   s is an integer ranging from 1 to 4; and    -   t is 0 or 1.

-   Additional Embodiment 31. The method of additional embodiment 30,    further comprising the step of detecting a total amount of target    within the sample.

-   Additional Embodiment 32. The method of additional embodiment 30,    wherein the first detection reagents include amplification    components to label the unmasked enzyme of the first target-unmasked    hapten-antibody conjugate complex with a plurality of first reporter    moieties.

-   Additional Embodiment 33. The method of additional embodiment 32,    wherein the plurality of first reporter moieties are haptens.

-   Additional Embodiment 34. The method of additional embodiment 33,    wherein the first detection reagents further comprise secondary    antibodies specific to the plurality of first reporter moieties,    each secondary antibody conjugated to a second reporter moiety.

-   Additional Embodiment 35. The method of additional embodiment 34,    wherein the second reporter moiety is selected from the group    consisting of an amplification enzyme or a fluorophore.

-   Additional Embodiment 36. The method of additional embodiment 34,    wherein the second reporter moiety is an amplification enzyme and    wherein the first detection reagents further comprise a first    chromogenic substrate or fluorescent substrate for the amplification    enzyme.

-   Additional Embodiment 37. The method of additional embodiment 30,    wherein the method further comprises a decaging step.

-   Additional Embodiment 38. A caged hapten having Formula (IE) or    (IF):

wherein

-   -   “Hapten” is a hapten;    -   Y is selected from a carbonyl-reactive group, an amine-reactive        group, or a thiol-reactive group;    -   A is a group comprising a branched or unbranched, substituted or        unsubstituted, saturated or unsaturated aliphatic group having        between 1 and 15 carbon atoms, and optionally having one or more        heteroatoms selected from the group consisting of O, N, or S;    -   X is a bond, or a group comprising a branched or unbranched,        substituted or unsubstituted, saturated or unsaturated aliphatic        group having between 1 and 30 carbon atoms, and optionally        having one or more heteroatoms selected from the group        consisting of O, N, or S;    -   W is a 5-, 6-, or 7-membered substituted or unsubstituted        aromatic or heterocyclic group;    -   each R¹ is independently selected from H, F, Cl, Br, I,        —O-methyl, —O— ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl,        —O-sec-butyl, or —O-iso-butyl; a —S-alkyl group having between 1        and 4 carbon atoms; an —O-alkyl group having between 1 and 4        carbon atoms; a —N(H)-alkyl group having between 1 and 4 carbon        atoms; a —N-(alkyl)₂ group having between 1 and 6 carbon atoms;        an alkyl group having between 1 and 4 carbon atoms and        optionally substituted with N or S; cyano groups; and carboxyl        groups;    -   V is a bond, a substituted or unsubstituted alkyl group having        between 1 and 4 carbon atoms, or a substituted or unsubstituted        —O-alkyl group;    -   R³ is an enzyme substrate;    -   q is 0 or 1;    -   s is 0 or an integer ranging from 1 to 4; and    -   t is 0 or 1.

-   Additional Embodiment 39. The caged hapten of additional embodiment    38, wherein the enzyme substrate is selected from the group    consisting of a phosphate group, an ester group, an amide group, a    sulfate group, a glycoside group, a urea group, and a nitro group.

-   Additional Embodiment 40. The caged hapten of additional embodiment    38, wherein each R¹ group is different.

-   Additional Embodiment 41. The caged hapten of additional embodiment    38, wherein X has the structure of Formula (IIIA):

wherein d and e are integers each independently ranging from 4 to 18; Qis a bond, O, S, or N(R^(c))(R^(d)); Ra and R^(b) are independently H, aC₁-C₄ alkyl group, F, Cl, or N(R^(c))(R^(d)); and R^(c) and R^(d) areindependently CH₃ or H. In some embodiments, d and e are integers eachindependently ranging from 1 to 24.

-   Additional Embodiment 42. The caged hapten of additional embodiment    38, wherein t is 1 and V is —CH₂—.-   Additional Embodiment 43. The caged hapten of additional embodiment    38, wherein t is 1 and V is —O—CH₂—.-   Additional Embodiment 44. The caged hapten of additional embodiment    38, wherein t is 1 and V is —C(R¹)₂ where R¹ is a C₁-C₄ alkyl group.-   Additional Embodiment 45. The caged hapten of additional embodiment    38, wherein t is 1 and V is —O—C(R¹)₂ where R¹ is a C₁-C₄ alkyl    group.-   Additional Embodiment 46. The caged hapten of additional embodiment    38, wherein s is 2, R¹ is —O—CH₃—, t is 1 and V is —CH₂—.

1. A compound having Formula (IE) or (IF):

wherein “Hapten” is a hapten; Y is selected from a carbonyl-reactive group, an amine-reactive group, or a thiol-reactive group; A is a group comprising a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having between 1 and 15 carbon atoms, and optionally having one or more heteroatoms selected from the group consisting of O, N, or S; X is a bond, or a group comprising a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having between 1 and 30 carbon atoms, and optionally having one or more heteroatoms selected from the group consisting of O, N, or S; W is a 5-, 6-, or 7-membered substituted or unsubstituted aromatic or heterocyclic group; each R¹ is independently selected from H, F, Cl, Br, I, —O-methyl, —O— ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl, —O-sec-butyl, or —O-iso-butyl; a —S-alkyl group having between 1 and 4 carbon atoms; an —O-alkyl group having between 1 and 4 carbon atoms; a —N(H)-alkyl group having between 1 and 4 carbon atoms; a —N-(alkyl)₂ group having between 1 and 6 carbon atoms; an alkyl group having between 1 and 4 carbon atoms and optionally substituted with N or S; cyano groups; and carboxyl groups; V is a bond, a substituted or unsubstituted alkyl group having between 1 and 4 carbon atoms, or a substituted or unsubstituted —O-alkyl group; R³ comprises an enzyme substrate; q is 0 or 1; s is 0 or an integer ranging from 1 to 4; and t is 0 or
 1. 2. The compound of claim 1, wherein the enzyme substrate is selected from the group consisting of a phosphate group, an ester group, an amide group, a sulfate group, a glycoside group, a urea group, and a nitro group.
 3. The compound of claim 1, wherein s ranges from 1 to 4, and wherein each R¹ group is different.
 4. The compound of claim 1, wherein X has the structure of Formula (IIIA):

wherein d and e are integers each independently ranging from 4 to 18; Q is a bond, O, S, or N(R^(c))(R^(d)); Ra and R^(b) are independently H, a C₁-C₄ alkyl group, F, Cl, or N(R^(c))(R^(d)); and R^(c) and R^(d) are independently CH₃ or H.
 5. The compound of claim 1, wherein s is
 2. 6. The compound of claim 5, wherein at least one R¹ group is —O—CH₃.
 7. The compound of claim 6, wherein t is 1; and V is —O—CH₂— or —CH₂—.
 8. The compound of claim 1, wherein when V is a substituted-alkyl group or a substituted —O-alkyl group, the -alkyl or —O-alkyl group is substituted with one or more an electron withdrawing groups.
 9. A compound having Formula (IE) or (IF):

wherein “Hapten” is one of a pyrazole, a nitropyrazole, a benzofuran, an oxazole, an oxazole sulfonamide, a thiazole, a thiazole sulfonamide, and derivatives or analogs thereof; Y is selected from a carbonyl-reactive group, an amine-reactive group, or a thiol-reactive group; A is a group comprising a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having between 1 and 15 carbon atoms, and optionally having one or more heteroatoms selected from the group consisting of O, N, or S; X is a bond, or a group comprising a branched or unbranched, substituted or unsubstituted, saturated or unsaturated aliphatic group having between 1 and 30 carbon atoms, and optionally having one or more heteroatoms selected from the group consisting of O, N, or S; W is a 6-membered substituted or unsubstituted aromatic group; each R¹ is independently selected from H, F, Cl, Br, I, —O-methyl, —O— ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl, —O-sec-butyl, or —O-iso-butyl; a —S-alkyl group having between 1 and 4 carbon atoms; a —N(H)-alkyl group having between 1 and 4 carbon atoms; V is a bond, a substituted or unsubstituted alkyl group having between 1 and 4 carbon atoms, or a substituted or unsubstituted —O-alkyl group; R³ comprises a phosphate group, an ester group, a sulfate group, a glycoside group, an amide group, a urea group, and a nitro group; q is 0 or 1; s is 0 or an integer ranging from 1 to 4; and t is 0 or
 1. 10. The compound of claim 9, wherein X has the structure of Formula (IIIA):

wherein d and e are integers each independently ranging from 4 to 18; Q is a bond, O, S, or N(R^(c))(R^(d)); Ra and R^(b) are independently H, a C₁-C₄ alkyl group, F, Cl, or N(R^(c))(R^(d)); and R^(c) and R^(d) are independently CH₃ or H.
 11. The compound of claim 9, wherein s is
 2. 12. The compound of claim 11, wherein at least one R¹ group is —O—CH₃.
 13. The compound of claim 12, wherein t is 1; and V is —O—CH₂— or —CH₂—.
 14. A compound having Formula (IE) or (IF):

wherein “Hapten” is a pyrazole or a derivative or analog thereof; Y is selected from a carbonyl-reactive group, an amine-reactive group, or a thiol-reactive group; A is a group comprising a branched or unbranched, unsubstituted aliphatic group having between 1 and 4 carbon atoms; X is an unsubstituted, saturated or unsaturated aliphatic group having between 1 and 8 carbon atoms, and optionally having one or more heteroatoms selected from the group consisting of O, N, or S; W is a 6-membered substituted aromatic group; each R¹ is independently selected from H, F, Cl, Br, I, —O-methyl, —O— ethyl, —O-n-propyl, —O-iso-propyl; —O-n-butyl, —O-sec-butyl, or —O-iso-butyl; V is a substituted or unsubstituted —O-alkyl group; R³ comprises a phosphate group, an ester group, a sulfate group, a glycoside group, an amide group, a urea group, and a nitro group; q is 0 or 1; s is an integer ranging from 1 to 4; and t is.
 15. The compound of claim 14, wherein s ranges from 1 to 4, and wherein each R¹ group is different.
 16. The compound of claim 14, wherein X has the structure of Formula (IIIA):

wherein d and e are integers each independently ranging from 4 to 8; Q is a bond, O, S, or N(R^(c))(R^(d)); Ra and R^(b) are independently H or a C₁-C₄ alkyl group.
 17. The compound of claim 14, wherein s is
 2. 18. The compound of claim 17, wherein at least one R¹ group is —O—CH₃.
 19. The compound of claim 18, wherein t is 1; and V is —O—CH₂— or —CH₂—.
 20. The caged hapten of claim 14, wherein the enzyme substrate comprises a phosphate group. 